Addressable flow cell using patterned electrodes

ABSTRACT

Disclosed are methods and systems concerning flow cells for sequencing a nucleic acid sample that may be characterized by the following components: a substrate having an inner surface facing a library sequencing region, and an outer surface; a plurality of a plurality of forward and reverse amplification primers immobilized over the inner surface and providing a nucleic acid library capture surface of the library sequencing region; a plurality of electrodes disposed along the inner surface directly under at least some of the forward and reverse amplification primers, and configured to provide, when charged, an electric field through the library capture surface and into the library sequencing region; electrical leads connected to the plurality of electrodes to permit the electrodes to be independently addressable; and fluidic couplings configured to deliver a plurality of nucleic acid libraries to the flow cell during different time periods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/107,882, filed on Jun. 23, 2016, which is a U.S. National Stage entryof PCT Application No. PCT/US2014/072699, filed on Dec. 30, 2014, whichclaims the benefit of and priority to U.S. Provisional Application No.61/922,604, filed Dec. 31, 2013, the contents of which are incorporatedherein by reference in their entirety and for all purposes.

INCORPORATION BY REFERENCE

All documents cited are, in relevant part, incorporated herein byreference in their entireties for the purposes indicated by the contextof their citation herein. However, the citation of any document is notto be construed as an admission that it is prior art with respect to thepresent disclosure.

BACKGROUND

The study of biology has recently benefited from improved methods ofanalysis and sequencing of nucleic acids. While the “human genome” hasbeen sequenced there are still vast amounts of genomic material toanalyze, e.g., genetic variation between different individuals, tissues,additional species, etc.

Devices for DNA sequencing based on separation of fragments of differinglength were first developed in the 1980s, and have been commerciallyavailable for a number of years. A number of new DNA sequencingtechnologies are based on the massively parallel analysis of unamplified(WO00006770; Proceedings of the National Academy of Sciences U.S.A, 100,3960-3964 (2003)) or amplified single molecules, either in the form ofplanar arrays (WO9844151) or on beads (WO04069849; Nature, 437, 376-380(2005); Science, 309, 5741, 1728-1732 (2005); Nat Biotechnol. 6,630-6344 (2000)).

The methodology used to analyze the sequence of the nucleic acids insuch new sequencing techniques is often based on the detection offluorescent nucleotides or oligonucleotides. The detectioninstrumentation used to read the fluorescence signals on such arrays maybe based on either epifluorescence or total internal reflectionmicroscopy, for example as described in WO9641011, WO00006770 orWO02072892.

Multiplexing enables large sample numbers to be simultaneously sequencedduring a single experiment. Some methodologies utilize individual“barcode” sequences that are added to each sample so that they may bedifferentiated during data analysis.

SUMMARY

Certain disclosed embodiments concern flow cells for sequencing anucleic acid sample that may be characterized by the followingcomponents: (a) a substrate having an inner surface facing a librarysequencing region, and an outer surface; (b) a plurality of a pluralityof forward and reverse amplification primers immobilized over the innersurface and providing a nucleic acid library capture surface of thelibrary sequencing region; (c) a plurality of electrodes disposed alongthe inner surface directly under at least some of the forward andreverse amplification primers, and configured to provide, when charged,an electric field through the library capture surface and into thelibrary sequencing region; (d) electrical leads connected to theplurality of electrodes to permit the electrodes to be independentlyaddressable; and (e) fluidic couplings configured to deliver a pluralityof nucleic acid libraries to the flow cell during different timeperiods.

In some implementations, the flow cell further includes one or moreseparation structures disposed over the inner surface and definingmultiple substantially parallel lanes on the library sequencing region.In some implementations, the fluidic couplings are configured deliverthe nucleic acid libraries to distinct lanes.

In some implementations, the flow cell further includes a cover arrangedsubstantially parallel to and spaced apart from the inner surface of thesubstrate, wherein the cover defines a boundary of the librarysequencing region. In some implementations, the cover is substantiallytransmissive to radiation of wavelengths suitable for imaging.

In some implementations, the cover includes a film of transparentelectrically conductive film attached to an electrical lead.

In some implementations, the library capture surface of the attachmentlayer occupies an area of between about 60 mm² and about 2400 mm².

In some implementations, the density of the plurality of forward andreverse amplification primers attached to the attachment layer is atleast 1 fmol per mm². In some implementations, the density of thehybridized polynucleotides attached to the solid support is 10,000/mm²to 2,000,000/mm².

In some implementations, the flow cell further includes an attachmentlayer to which the plurality of forward and reverse amplificationprimers is attached.

In some implementations, the controller is designed or programmed toapply the positive charge to the first electrode at a voltage in therange of approximately 0.5-3V. In some implementations, the controlleris designed or programmed to apply the positive charge to the secondelectrode at a voltage in the range of approximately 0.5-3V. In someimplementations, the controller is designed or programmed to apply thepositive charge to the first electrode to produce a current in the rangeof approximately 250 nA-5 μA. In some implementations, the controller isdesigned or programmed to apply the positive charge to the secondelectrode to produce a current in the range of approximately 250 nA-5μA. In some implementations, the controller is designed or programmed toapply the positive charge to the first electrode to produce an electricfield in the range of approximately 10-200 V/cm. In someimplementations, the controller is designed or programmed to apply thepositive charge to the second electrode to produce an electric field inthe range of approximately 10-200 V/cm. In some implementations, thecontroller is designed or programmed to operate the first and secondelectrodes in direct current (DC) mode. In some implementations, thecontroller is designed or programmed to apply the positive charge to thesecond electrode using direct current (DC).

In some implementations, the electrodes are disposed perpendicular tothe length of the solid support. In some implementations, the electrodesare disposed in an array. In some implementations, the plurality ofelectrodes includes about 2-12 electrodes disposed along the solidsupport. In some implementations, each of the plurality of electrodeshas a surface area of approximately 100 um² to 200 mm². In someimplementations, each of the plurality of electrodes is substantiallyround-shaped. In some implementations, the electrodes are made of gold.

In some implementations, the plurality of electrodes are made ofindium-doped tin oxide (ITO). In other implementations, the plurality ofelectrodes are made of a conductor selected from the group consisting ofsilver, tin, titanium, copper, platinum, palladium, polysilicon, andcarbon. In some implementations, the conductance of each of theplurality of electrodes is in the range of approximately 280 nS-1 μS.

In some implementations, the flow cell further includes a controllerdesigned or programmed to deliver electrical charge to each of theelectrodes independently. In some implementations, the controller isfurther designed or programmed to deliver a positive charge to oneelectrode while delivering negative charge to a plurality of adjacentelectrodes. In some implementations, the controller is further designedor programmed to control delivery of the different nucleic acidlibraries to the flow cell at times corresponding to delivery ofpositive charge to different electrodes.

In some implementations, the forward and reverse amplification primersare configured to hybridize to specific gene sequences of the nucleicacid libraries. In some implementations, the specific gene sequences areone of: a barcode region of the nucleic acid libraries, an adapterregion of the nucleic acid libraries, and nucleic acid sequences ofinterest within the nucleic acid libraries.

In some implementations, amplification primers configured to hybridizeto a first gene sequence are localized in a first region of the flowcell, and wherein amplification primers configured to hybridize to asecond gene sequence are localized to a second, spatially separate,region of the flow cell. In some implementations, the amplificationprimers are localized to the first and second regions using electricfields generated by the plurality of electrodes. In someimplementations, the amplification primers are disposed on one or morebeads immobilized on the inner surface of the substrate. In someimplementations, the amplification primers are disposed on one or morebeads immobilized in one or more wells of the inner surface of thesubstrate.

Certain disclosed embodiments concern methods of sequencing nucleic acidsamples that may be characterized by the following operations: (a)introducing a first nucleic acid library to a library sequencing regionof a flow cell including: a substrate having an inner surface facing thelibrary sequencing region, and an outer surface, a plurality of forwardand reverse amplification primers disposed over the inner surface andproviding a nucleic acid library capture surface of the librarysequencing region, and a plurality of electrodes disposed along thesubstrate proximate the library capture surface; (b) applying a positivecharge to a first set of one or more electrodes, from the plurality ofelectrodes, while the first nucleic acid library flows through thelibrary sequencing region to thereby attract nucleic acids from thefirst nucleic acid library to the forward and reverse amplificationprimers disposed proximate the first set of one or more electrodes suchthat members of the first nucleic acid library hybridize to forward andreverse amplification primers proximate the first set of one or moreelectrodes, wherein members of the first nucleic acid library do notsubstantially hybridize to forward and reverse amplification primerslocated proximate electrodes that do not have positive charge applied;(c) introducing a second nucleic acid library to a library sequencingregion flow cell; and (d) applying a positive charge to a second set ofone or more electrodes, from the plurality of electrodes, while thesecond nucleic acid library flows through the library sequencing regionto thereby attract nucleic acids from the second nucleic acid library tothe forward and reverse amplification primers disposed proximate thesecond set of one or more electrodes such that members of the secondnucleic acid library hybridize to forward and reverse amplificationprimers proximate the second set of one or more electrodes, whereinmembers of the second nucleic acid library do not substantiallyhybridize to forward and reverse amplification primers located proximateelectrodes that do not have positive charge applied.

In some implementations, introducing the first nucleic acid library andintroducing the second nucleic acid library includes introducing thefirst and second nucleic acid libraries to the same lane of the flowcell.

In some implementations, the method further includes applying negativecharge to electrodes adjacent to the first electrode while applying thepositive charge to the first electrode.

In some implementations, the method further includes (e) introducing athird nucleic acid library to a library sequencing region flow cell and(f) applying a positive charge to a third set of one or more electrodes,from the plurality of electrodes, while the third nucleic acid libraryflows through the library sequencing region to thereby attract nucleicacids from the third nucleic acid library to the forward and reverseamplification primers disposed proximate the third set of one or moreelectrodes such that members of the third nucleic acid library hybridizeto forward and reverse amplification primers proximate the third set ofone or more electrodes, wherein members of the third nucleic acidlibrary do not substantially hybridize to forward and reverseamplification primers located proximate electrodes that do not havepositive charge applied.

In some implementations, the method further includes, when applying apositive charge to a first electrode, applying a positive charge tothird electrode from among the plurality of electrodes, which thirdelectrode is not the second electrode, such that members of the firstnucleic acid library hybridize to forward and reverse amplificationprimers proximate the other electrode.

In some implementations, introducing the first nucleic acid libraryincludes flowing the first nucleic acid library in a solution includingone or more protective reagents that blocks detrimental effects of waterelectrolysis.

In some implementations, the one or more protective reagents undergoes aredox reaction at an electric potential that is below the electricpotential at which water electrolyzes, and wherein the redox reactionproduces only products that are substantially benign to nucleic acids.In some implementations, the one or more protective reagents is selectedfrom the group consisting of α-thioglycerol, dithiothreitol,hydroquinone, ferrocyanide, and β-mercaptoethanol. In someimplementations, the one or more protective reagents is present in thesolution at a concentration of about 25 mM-1.5 M.

In some implementations, the method further includes preparing the firstnucleic acid library by: fragmenting a complex polynucleotide sample togenerate a plurality of target polynucleotide fragments; and ligatingidentical mismatched adapter polynucleotides to both ends of each of thedifferent target polynucleotide fragments to form adapter-targetconstructs, wherein each mismatched adapter is formed from two annealedpolynucleotide strands that form a bimolecular complex including atleast one double-stranded region and a mismatched region includingportions of both strands, wherein the ligating covalently attaches eachstrand of the at least one double-stranded region to each respectivestrand of each of the different target polynucleotide fragment togenerate adapter-target constructs including covalently attached 5′ and3′ adapter sequences.

In some implementations, each hybridized nucleic acid is amplified by:forming at least one nucleic acid template including the at least onenucleic acid to be amplified, wherein the at least one nucleic acidcontains an oligonucleotide sequence Y at the 5′ end and anoligonucleotide sequence Z at the 3′ end, and the at least one nucleicacid carries a means for immobilizing the at least one nucleic acid to asolid support at the 5′ end; mixing the at least one nucleic acidtemplate, in the presence of the solid support, with one or more colonyprimers X, each of which can hybridize to the oligonucleotide sequence Zand carries a means for immobilizing the colony primer to the solidsupport at the 5′ end, whereby the 5′ ends of both the at least onenucleic acid template and the colony primers are immobilized to thesolid support, wherein the 5′ ends of both the at least one nucleic acidtemplate and the colony primers are immobilized to the solid supportsuch that they cannot be removed by washing with water or aqueous bufferunder DNA denaturing conditions; and performing one or more nucleic acidamplification reactions on the immobilized nucleic acid template, sothat nucleic acid colonies are generated.

In some implementations, the method further includes: moving one or morefluorescently labeled reagents through the flow cell into contact withthe hybridized members of the first and second nucleic acid libraries,wherein the reagents include components to extend a second sequencecomplementary to the hybridized polynucleotides; illuminating thehybridized polynucleotides with at least one excitation laser coupledthrough a fiberoptic device; detecting, using at least onecharge-coupled device (CCD) camera, fluorescence emissions of thefluorescently labeled reagents; and determining, based on thefluorescence emissions, an identity of the second sequence.

In some implementations, the forward and reverse amplification primersare configured to hybridize to specific gene sequences of the nucleicacid libraries. In some implementations, the specific gene sequences areone of: a barcode region of the nucleic acid libraries, an adapterregion of the nucleic acid libraries, and nucleic acid sequences ofinterest within the nucleic acid libraries. In some implementations,amplification primers configured to hybridize to a first gene sequenceare localized in a first region of the flow cell, and whereinamplification primers configured to hybridize to a second gene sequenceare localized to a second, spatially separate, region of the flow cell.In some implementations, the amplification primers are localized to thefirst and second regions using electric fields generated by theplurality of electrodes. In some implementations, the amplificationprimers are disposed on one or more beads immobilized on the innersurface of the substrate. In some implementations, the amplificationprimers are disposed on one or more beads immobilized in one or morewells of the inner surface of the substrate.

Certain disclosed embodiments concern systems for sequencingpolynucleotide samples that may be characterized by the followingcomponents: (a) a solid support having a plurality of electrodesdisposed thereon, the solid support including an attachment layer overthe plurality of electrodes, the attachment layer having a librarycapture surface including a plurality of forward and reverseamplification primers immobilized thereon; (b) electrical leadsconnected to the plurality of electrodes to permit the electrodes to beindependently addressable; (c) a fluid direction system for controllablydelivering a plurality of polynucleotide libraries in a buffer with areducing agent to the library capture surface during different timeperiods; and (d) a controller for controlling the fluid direction systemand for delivering current and/or potential to the electrodes, whereinthe controller is designed or programmed to apply a positive charge to afirst electrode of the plurality of electrodes while the fluid directionsystem delivers a first polynucleotide library along the solid supportsuch that polynucleotides from the first polynucleotide library areattracted to forward and reverse amplification primers disposedproximate the first electrode such that members of the firstpolynucleotide library hybridize to forward and reverse amplificationprimers proximate the first electrode, wherein members of the firstpolynucleotide library do not substantially hybridize to forward andreverse amplification primers located proximate electrodes that do nothave positive charge applied, and apply a positive charge to a secondelectrode of the plurality of electrodes while the fluid directionsystem delivers a second polynucleotide library along the solid supportsuch that polynucleotides from the second polynucleotide library areattracted to forward and reverse amplification primers disposedproximate the second electrode such that members of the secondpolynucleotide library hybridize to forward and reverse amplificationprimers proximate the second electrode, wherein members of the secondpolynucleotide library do not substantially hybridize to forward andreverse amplification primers located proximate electrodes that do nothave positive charge applied.

In some implementations, the solid support is provided in a flow cellincluding one or more fluidic channels in which the forward and reverseamplification primers are attached, the one or more fluidic channelsdefining multiple substantially parallel lanes on the library capturesurface. In some implementations, the controller is further designed orprogrammed to cause the fluid direction system to deliver thepolynucleotide libraries to distinct lanes of the flow cell.

In some implementations, the system further includes a coversubstantially parallel to the solid support and spaced apart from theinner surface, wherein the cover defines a boundary of the librarycapture surface. In some implementations, the cover is substantiallytransmissive to radiation of wavelengths suitable for imaging. In someimplementations, the cover includes a transparent electricallyconductive layer attached to an electrical lead.

In some implementations, the conductive layer includes indium-doped tinoxide (ITO).

In some implementations, the library capture surface of the attachmentlayer occupies an area of between about 60 mm² and about 2400 mm².

In some implementations, the density of the plurality of forward andreverse amplification primers attached to the attachment layer is atleast 1 fmol per mm². In some implementations, the density of thehybridized polynucleotides attached to the solid support is 10,000/mm²to 2,000,000/mm². In some implementations, the system further includesan attachment layer to which the plurality of forward and reverseamplification primers are attached.

In some implementations, the controller is designed or programmed toapply the positive charge to the first electrode at a voltage in therange of approximately 0.5-3V. In some implementations, the controlleris designed or programmed to apply the positive charge to the secondelectrode at a voltage in the range of approximately 0.5-3V. In someimplementations, the controller is designed or programmed to apply thepositive charge to the first electrode to produce a current in the rangeof approximately 250 nA-5 μA. In some implementations, the controller isdesigned or programmed to apply the positive charge to the secondelectrode to produce a current in the range of approximately 250 nA-5μA. In some implementations, the controller is designed or programmed toapply the positive charge to the first electrode to produce an electricfield in the range of approximately 10-200 V/cm. In someimplementations, the controller is designed or programmed to apply thepositive charge to the second electrode to produce an electric field inthe range of approximately 10-200 V/cm. In some implementations, thecontroller is designed or programmed to operate the first and secondelectrodes in direct current (DC) mode. In some implementations, thecontroller is designed or programmed to apply the positive charge to thesecond electrode using direct current (DC).

In some implementations, the electrodes are disposed perpendicular tothe length of the solid support. In some implementations, the electrodesare disposed in an array. In some implementations, the plurality ofelectrodes includes about 2-12 electrodes are disposed along the solidsupport. In some implementations, each of the plurality of electrodeshas a surface area of approximately 100 um² to 200 mm². In someimplementations, each of the plurality of electrodes is substantiallyround-shaped. In some implementations, the electrodes are made of gold.In other implementations, the plurality of electrodes is made ofindium-doped tin oxide (ITO).

In other implementations, the plurality of electrodes is made of aconductor selected from the group consisting of silver, tin, titanium,copper, platinum, palladium, polysilicon, and carbon. In someimplementations, the conductance of each of the plurality of electrodesis in the range of approximately 280 nS-1 μS.

In some implementations, the controller is further designed orprogrammed to deliver a positive charge to one electrode whiledelivering negative charge to a plurality of adjacent electrodes. Insome implementations, the controller is further designed or programmedto control delivery of the different nucleic acid libraries to the flowcell at times corresponding to delivery of positive charge to differentelectrodes.

In some implementations, the forward and reverse amplification primersare configured to hybridize to specific gene sequences of thepolynucleotide libraries. In some implementations, the specific genesequences are one of: a barcode region of the polynucleotide libraries,an adapter region of the polynucleotide libraries, and polynucleotidesequences of interest within the polynucleotide libraries. In someimplementations, amplification primers configured to hybridize to afirst gene sequence are localized in a first region of the flow cell,and wherein amplification primers configured to hybridize to a secondgene sequence are localized to a second, spatially separate, region ofthe flow cell. In some implementations, the amplification primers arelocalized to the first and second regions using electric fieldsgenerated by the plurality of electrodes. In some implementations, theamplification primers are disposed on one or more beads immobilized onthe library capture surface. In some implementations, the amplificationprimers are disposed on one or more beads immobilized in one or morewells of the library capture surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a portion of a flow cell including two electrodes disposedon a solid support and an attachment layer, in accordance with someimplementations.

FIG. 1B shows a top and side view of a lane of a flow cell including sixelectrodes disposed on the flow cell, in accordance with someimplementations.

FIG. 2A shows a portion of a flow cell including two electrodes disposedon a solid support and an attachment layer, in accordance with someimplementations.

FIG. 2B shows a top and side view of a lane of a flow cell including sixelectrodes disposed on the flow cell, in accordance with someimplementations.

FIG. 2C shows a top and side view of a lane of a flow cell including sixelectrodes disposed on the flow cell, in accordance with someimplementations.

FIG. 3A shows a portion of a flow cell including two electrodes disposedon a solid support and an attachment layer, in accordance with someimplementations.

FIG. 3B shows a top and side view of a lane of a flow cell including sixelectrodes disposed on the flow cell, in accordance with someimplementations.

FIG. 3C shows a top and side view of a lane of a flow cell including sixelectrodes disposed on the flow cell, in accordance with someimplementations.

FIG. 3D shows a flow chart of a process of sequentially flowing one ormore libraries through a library sequencing region while activatingdifferent electrodes for multiplexed sequencing of the one or morelibraries, in accordance with some implementations.

FIG. 4 shows the percentage of transported DNA that is captured whenelectrodes are used to capture the DNA, in accordance with someimplementations.

FIG. 5 shows the electric field distribution along the length and heightof the flow cell when an electrode is positively charged, in accordancewith some implementations.

FIG. 6 is an exploded view of a flow cell having eight lanes and eightelectrodes disposed along the bottom plate of the flow cell, inaccordance with some implementations.

FIG. 7 shows a flow cell used for in-line sample preparation usingimmobilized Tn5 transposons, in accordance with some implementations.

FIG. 8 shows the current to voltage relationship in the flow cell andthe point at which electrolysis occurs, in accordance with someimplementations.

FIG. 9 shows the effect of including a reducing agent (2-mercap) in thebuffer on the extent to which DNA clusters form over the electrodes, inaccordance with some implementations.

FIG. 10A shows a flow chart of a process of seeding a DNA library whilea set of electrodes is positively charged, followed by reversing thebias of the set of electrodes, to demonstrate the extent ofcross-contamination of clusters on the other set of electrodes, inaccordance with some implementations.

FIG. 10B demonstrates the amount of cross-contamination resulting fromthe process described in FIG. 10A, in accordance with someimplementations.

FIG. 11A shows the voltage at which polynucleotides will concentrateover the electrodes when Indium tin oxide (ITO) electrodes are used, inaccordance with some implementations.

FIG. 11B shows the voltage at which polynucleotides will concentrateover the electrodes when ITO electrodes are used, in accordance withsome implementations.

FIG. 12A shows the voltage at which polynucleotides will concentrateover the electrodes when ITO electrodes are used and when 250 mMhydroquinone is added to the buffer, in accordance with someimplementations.

FIG. 12B shows the current to voltage relationship when ITO electrodesare used in a buffer containing 10% 2-mercaptoethanol and in a buffernot containing any 2-mercaptoethanol, in accordance with someimplementations.

FIG. 12C shows the current to voltage relationship when ITO electrodesare used in a buffer containing hydroquinone, in accordance with someimplementations.

FIGS. 13A and 13B display an exemplary embodiment of a flow cell.

FIGS. 13C and 13D display additional exemplary flow cell designs.

FIG. 14 is an exploded view of a flow cell having eight lanes and eightelectrodes disposed along the bottom plate of the flow cell.

FIG. 15A shows a planar view of two possible configurations ofelectrodes on a flow cell.

FIG. 15B shows a side view of two possible configurations of electrodeson a flow cell.

FIGS. 16A and 16B show an example of a design where the gold electrodesare partially covered and passivated by silicon nitride.

FIG. 17 shows an exemplary TIRF imaging configuration of a backlightdesign embodiment of a sequencing system.

FIG. 18 shows an exemplary arrangement of a sequencing system of thedisclosed embodiments.

FIGS. 19A-C present generalized diagrams of exemplary fluid flowarrangements of some embodiments.

FIG. 20 shows how the electrodes of the flow cell may be interfaced withthe sequencing system and controller.

FIG. 21 shows an exemplary configuration of two sets of electrodescontrolled by two voltage sources.

FIG. 22 shows a flow chart of a process of sequentially seeding twolibraries while holding the two sets of electrodes at different charges,and the imaging results of the polynucleotide hybridization.

DETAILED DESCRIPTION Definitions

Before describing some of the embodiments in detail, it is to beunderstood that the invention herein is not limited to use with anyparticular nucleic acids or biological systems. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aflow cell” optionally includes a combination of two or more flow cells,and the like.

As used herein, the terms “polynucleotide”, “nucleic acid” and “nucleicacid molecules” are used interchangeably and refer to a covalentlylinked sequence of nucleotides (i.e., ribonucleotides for RNA anddeoxyribonucleotides for DNA) in which the 3′ position of the pentose ofone nucleotide is joined by a phosphodiester group to the 5′ position ofthe pentose of the next, include sequences of any form of nucleic acid,including, but not limited to RNA and DNA molecules. The term“polynucleotide” includes, without limitation, single- anddouble-stranded polynucleotide. The terms should be understood toinclude, as equivalents, analogs of either DNA or RNA made fromnucleotide analogs. The terms as used herein also encompass cDNA, thatis complementary, or copy, DNA produced from an RNA template, forexample by the action of reverse transcriptase. The terms nucleic acid,polynucleotide and oligonucleotide are not intended to denote anyparticular difference in size, sequence, or other property unlessspecifically indicated otherwise. For clarity of description the termsmay be used to distinguish one species of molecule from another whendescribing a particular method or composition that includes severalmolecular species.

The single stranded polynucleotide molecules sequenced by the systemsand devices herein can have originated in single-stranded form, as DNAor RNA or have originated in double-stranded DNA (dsDNA) form (e.g.genomic DNA fragments, PCR and amplification products and the like).Thus a single stranded polynucleotide may be the sense or antisensestrand of a polynucleotide duplex. Methods of preparation of singlestranded polynucleotide molecules suitable for use in the describedmethods using standard techniques are well known in the art. The precisesequence of the primary polynucleotide molecules is generally notmaterial to the disclosed embodiments and may be known or unknown. Thesingle stranded polynucleotide molecules can represent genomic DNAmolecules (e.g., human genomic DNA) including both intron and exonsequences (coding sequence), as well as non-coding regulatory sequencessuch as promoter and enhancer sequences.

In certain embodiments, a nucleic acid contains a “target” region thatit is desired to fully or partially sequence. The nature of the targetregion is not limiting to the disclosed embodiments. It may be of apreviously known or unknown sequence and may be derived, for example,from a genomic DNA fragment, a cDNA, etc. The nucleic acid molecule mayalso include non-target sequences, for example at the 5′ and 3′ endsflanking the target region. If the nucleic acid is formed by solid-phasenucleic acid amplification, these non-target sequences may be derivedfrom the primers used for the amplification reaction. Sites for cleavageof one or both strands of a double-stranded nucleic acid may bepositioned in the non-target sequences.

The nucleic acid may form part of a cluster or colony comprised of manysuch nucleic acid molecules, and the cluster or colony may itself formpart of an array of such clusters or colonies, referred to herein as a“clustered array”. On such an array each nucleic acid molecule withineach colony will comprise the same target region, whereas differentcolonies may be formed of nucleic acid molecules comprising differenttarget regions. In certain embodiments, at least 90%, more preferably atleast 95%, of the colonies on a given clustered array will be formedfrom nucleic acid molecules comprising different target regions,although within each individual colony on the array all nucleic acidmolecules will contain the same target region.

As used throughout, the term “target nucleic acid” can be any moleculeto be selected and, optionally, amplified or sequenced. Target nucleicacids for use in the provided methods may be obtained from anybiological sample using known, routine methods. Suitable biologicalsamples include, but are not limited to, a blood sample, biopsyspecimen, tissue explant, organ culture, biological fluid or any othertissue or cell preparation, or fraction or derivative thereof orisolated therefrom. The biological sample can be a primary cell cultureor culture adapted cell line including but not limited to geneticallyengineered cell lines that may contain chromosomally integrated orepisomal recombinant nucleic acid sequences, immortalized orimmortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,stem cells, germ cells (e.g. sperm, oocytes), transformed cell lines andthe like. For example, polynucleotide molecules may be obtained fromprimary cells, cell lines, freshly isolated cells or tissues, frozencells or tissues, paraffin embedded cells or tissues, fixed cells ortissues, and/or laser dissected cells or tissues. Biological samples canbe obtained from any subject or biological source including, forexample, human or non-human animals, including mammals and non-mammals,vertebrates and invertebrates, and may also be any multicellularorganism or single-celled organism such as a eukaryotic (includingplants and algae) or prokaryotic organism, archaeon, microorganisms(e.g. bacteria, archaea, fungi, protists, viruses), and aquaticplankton.

The target nucleic acid described herein can be of any length suitablefor use in the provided methods. For example, the target nucleic acidscan be at least 10, at least 20, at least 30, at least 40, at least 50,at least 75, at least 100, at least 150, at least 200, at least 250, atleast 500, or at least 1000 nucleotides in length or longer. Generally,if the target nucleic acid is a small RNA molecule, the target nucleicacid will be at least 10 nucleotides in length. Thus, the target nucleicacid sequences can comprise RNA molecules, for example, small RNAmolecules including, but not limited to miRNA molecules, siRNAmolecules, tRNA molecules, rRNA molecules, and combinations thereof. Insome embodiments, the target nucleic acid sequence comprisessingle-stranded DNA.

In certain embodiments, the nucleic acid to be sequenced through use ofthe disclosed embodiments is immobilized upon a substrate (e.g., asubstrate within a flow cell or one or more beads upon a substrate suchas a flow cell, etc.). The term “immobilized” or “attached” as usedherein is intended to encompass direct or indirect, covalent ornon-covalent attachment, unless indicated otherwise, either explicitlyor by context. In certain embodiments, covalent attachment may be used,but generally all that is required is that the molecules (for example,nucleic acids) remain immobilized or attached to a support underconditions in which it is intended to use the support, for example inapplications requiring nucleic acid amplification and/or sequencing.Typically oligonucleotides to be used as capture oligonucleotides oramplification oligonucleotides are immobilized such that a 3′ end isavailable for enzymatic extension and at least a portion of the sequenceis capable of hybridizing to a complementary sequence. Immobilizationcan occur via hybridization to a surface attached oligonucleotide, inwhich case the immobilized oligonucleotide or polynucleotide may be inthe 3′-5′ orientation. Alternatively, immobilization can occur by meansother than base-pairing hybridization, such as the covalent attachmentset forth above.

The term “library” refers to a collection or plurality of nucleic acidtemplate molecules which have a common use or common property such as acommon origin; e.g., all members of the library come from a singlesample. The members of the library may be processed or modified to sothat their membership in the library is clearly identified. For example,all members of a library may share a common sequence at their 5′ endsand a common sequence at their 3′ ends. Use of the term “library” torefer to a collection or plurality of template molecules should not betaken to imply that the templates making up the library are derived froma particular source, or that the “library” has a particular composition.By way of example, use of the term “library” should not be taken toimply that the individual templates within the library must be ofdifferent nucleotide sequence or that the templates be related in termsof sequence and/or source.

As used throughout, “primers” and “amplification primers” are usedinterchangeably and are oligonucleotide sequences that are capable ofannealing specifically to a polynucleotide sequence to be amplifiedunder conditions encountered in a primer annealing step of anamplification reaction.

The term “solid support” (or “substrate” in certain usages) as usedherein refers to any inert substrate or matrix to which nucleic acidscan be attached, such as for example glass surfaces, plastic surfaces,latex, dextran, polystyrene surfaces, polypropylene surfaces,polyacrylamide gels, gold surfaces, and silicon wafers. In manyembodiments, the solid support is a glass surface (e.g., the planarsurface of a flow cell channel). The solid support may be mounted on theinterior of a flow cell to allow the interaction with solutions ofvarious reagents. In certain embodiments the solid support may comprisean inert substrate or matrix which has been “functionalized,” forexample by the application of a layer or coating of an intermediatematerial comprising reactive groups which permit covalent attachment tomolecules such as polynucleotides. By way of non-limiting example suchsupports can include polyacrylamide hydrogels supported on an inertsubstrate such as glass. In such embodiments the molecules(polynucleotides) can be directly covalently attached to theintermediate material (e.g. the hydrogel) but the intermediate materialcan itself be non-covalently attached to the substrate or matrix (e.g.the glass substrate). Covalent attachment to a solid support is to beinterpreted accordingly as encompassing this type of arrangement. Incertain embodiments, a solid support may contain a nucleic acid librarycapture surface having moieties for immobilizing target nucleic acidsthat come in contact with the substrate. The moieties may be a pluralityof primers or other capture moieties immobilized on a support surface.In one example, the primers are forward and reverse amplificationprimers.

As used herein, the term “interrogate” can refer to any interaction of amolecule on the array with any other chemical or molecule and may alsorefer to any analysis of a detectable signal from a molecule on thearray or any other molecule which is bound thereto or associatedtherewith. In one embodiment “interrogation” encompasses a targetpolynucleotide on the array functioning as a template upon which DNApolymerase acts. In other words, “interrogating” can encompasscontacting the target polynucleotides with another molecule, e.g., apolymerase, a nucleoside triphosphate, a complementary nucleic acidsequence, where the physical interaction provides information regardinga characteristic of the arrayed target polynucleotide. The contactingcan involve covalent or non-covalent interactions with the othermolecule. As used herein, “information regarding a characteristic” meansinformation about the identity or sequence of one or more nucleotides inthe target polynucleotide, the length of the polynucleotide, the basecomposition of the polynucleotide, the T_(m) of the polynucleotide, thepresence of a specific binding site for a polypeptide, a complementarynucleic acid or other molecule, the presence of an adduct or modifiednucleotide, or the three-dimensional structure of the polynucleotide.

The term “read” refers to a sequence read from a portion of a nucleicacid sample. Typically, though not necessarily, a read represents ashort sequence of contiguous base pairs in the sample. The read may berepresented symbolically by the base pair sequence (in ATCG) of thesample portion. It may be stored in a memory device and processed asappropriate to determine whether it matches a reference sequence ormeets other criteria. A read may be obtained directly from a sequencingapparatus or indirectly from stored sequence information concerning thesample. In some cases, a read is a DNA sequence of sufficient length(e.g., at least about 30 bp) that can be used to identify a largersequence or region, e.g. that can be aligned and specifically assignedto a chromosome or genomic region or gene.

As used herein, the terms “aligned”, “alignment”, or “aligning” refer tothe process of comparing a read or tag to a reference sequence andthereby determining whether the reference sequence contains the readsequence. If the reference sequence contains the read, the read may bemapped to the reference sequence or, in certain embodiments, to aparticular location in the reference sequence. In some cases, alignmentsimply tells whether or not a read is a member of a particular referencesequence (i.e., whether the read is present or absent in the referencesequence). For example, the alignment of a read to the referencesequence for human chromosome 13 will tell whether the read is presentin the reference sequence for chromosome 13. A tool that provides thisinformation may be called a set membership tester. In some cases, analignment additionally indicates a location in the reference sequencewhere the read or tag maps to. For example, if the reference sequence isthe whole human genome sequence, an alignment may indicate that a readis present on chromosome 13, and may further indicate that the read ison a particular strand and/or site of chromosome 13.

Aligned reads or tags are one or more sequences that are identified as amatch in terms of the order of their nucleic acid molecules to a knownsequence from a reference genome. Alignment can be done manually,although it is typically implemented by a computer algorithm, as itwould be impossible to align reads in a reasonable time period forimplementing the methods disclosed herein. One example of an algorithmfrom aligning sequences is the Efficient Local Alignment of NucleotideData (ELAND) computer program distributed as part of the Illumina®Genomics Analysis® pipeline. Alternatively, a Bloom filter or similarset membership tester may be employed to align reads to referencegenomes. See U.S. Patent Application No. 61/552,374 filed Oct. 27, 2011which is incorporated herein by reference in its entirety. The matchingof a sequence read in aligning can be a 100% sequence match or less than100% (non-perfect match).

As used herein, the term “reference genome” or “reference sequence”refers to any particular known genome sequence, whether partial orcomplete, of any organism or virus which may be used to referenceidentified sequences from a subject. For example, a reference genomeused for human subjects as well as many other organisms is found at theNational Center for Biotechnology Information at www.ncbi.nlm.nih.gov. A“genome” refers to the complete genetic information of an organism orvirus, expressed in nucleic acid sequences.

In various embodiments, the reference sequence is significantly largerthan the reads that are aligned to it. For example, it may be at leastabout 100 times larger, or at least about 1000 times larger, or at leastabout 10,000 times larger, or at least about 10⁵ times larger, or atleast about 10⁶ times larger, or at least about 10⁷ times larger.

In one example, the reference sequence is that of a full length humangenome. Such sequences may be referred to as genomic referencesequences. In another example, the reference sequence is limited to aspecific human chromosome such as chromosome 13. Such sequences may bereferred to as chromosome reference sequences. Other examples ofreference sequences include genomes of other species, as well aschromosomes, sub-chromosomal regions (such as strands), etc. of anyspecies. In various embodiments, the reference sequence is a consensussequence or other combination derived from multiple individuals.However, in certain applications, the reference sequence may be takenfrom a particular individual.

Introduction

The disclosed embodiments pertain to systems and devices to analyze alarge number of different nucleic acid sequences from, e.g., clonallyamplified single-molecule DNA arrays in flow cells, or arrays ofimmobilized beads. In particular, the systems and devices utilizeelectrode arrays configured to selectively capture and isolate specificnucleic acids on regions of a solid support. In various disclosedembodiments, the electrode arrays spatially isolate nucleic acidlibraries, so that one or more libraries are immobilized at one locationon a library capture surface while other libraries are immobilized atone or more other locations on the library capture surface. The membersof a nucleic acid library are captured on a region proximate anelectrode when a positive bias is selectively applied to the electrode.By bringing different libraries to the library capture surface atdifferent times, when different electrodes are activated by applying apositive charge, the system spatially isolates the nucleic acidlibraries from one another. In some implementations, different libraries(possibly from different samples) are captured at different regions of aflow cell for multiplexed sequencing. The regions are associated withspecific libraries. The nucleic acids in the different regions aresequenced in parallel but are identified by their libraries which areassociated with distinct regions, which have discrete electrodes.

The electrodes may be attached to or proximate a solid support having alibrary capture surface. The electrodes are independently addressable,such that one or more selected electrodes are given a positive chargewhile the remaining electrodes are given a negative or neutral charge.In this manner, a system may apply a spatially isolated electric fieldto a library capture surface and thereby selectively capture andspatially isolate a library on a region of the solid support. Aspatially and temporally controlled electric field applied in thismanner may be used in various sequencing platforms to increasesequencing throughput and sensitivity. Specifically, an addressableelectric field can isolate different nucleic samples into differentregions of a solid support such as a support used in sequencing flowcells or chips for multiplexed sequencing.

In some cases, an electric field may be employed to deliver forward-readprimers and reverse-read primers to different regions of the sequencingflow cells or chips for paired-end read in parallel, in which a forwardread is performed on polynucleotides hybridized to the forward-readprimers and a reverse read is performed on polynucleotides hybridized tothe reverse-read primers.

In some embodiments, the primers recognize an adapter region of thetarget library polynucleotides. In other embodiments, primers designedfor gene or other sequence-specific capture may recognize specificsequences of the target polynucleotides. Alternatively, the primers mayrecognize a barcode region of the target polynucleotides. In someembodiments, an electric field may be used to localize differentsequence-specific capture primers in specific regions of a flow cell,followed by hybridization of target polynucleotide libraries with orwithout an electric field.

In certain DNA sequencing technologies that utilize beads containingpolynucleotide libraries located in different regions of a chip formultiplexed sequencing, an electric field may be used to distributethose beads to their targeted locations. In some embodiments, the beadsmay be functionalized with primers and may be immobilized on flow cellsurface or wells or one or more electrodes, such that an electric fieldmay direct hybridization onto the immobilized beads.

In technologies that isolate polynucleotide templates in sensor wellsfor sequencing, an electric field may deliver forward-read andreverse-read primers into different sensor wells so that the forward andreverse reads may be performed in parallel, reducing the amount of timerequired for paired-end sequencing. In technologies that isolatesingle-stranded DNA templates in zero-mode waveguides on a chip, anelectric field may be used to distribute DNA libraries from differentsamples to different regions of the chip for multiplexed sequencing.

The various methods, apparatus, systems and uses are described infurther detail in the following examples which are not in any wayintended to limit the scope of the disclosure. Many of the embodimentspertain to sequencing by synthesis technology that generates clustersfrom sample DNA captured by paired end primers on a solid support. Thoseof skill in the art will understand that numerous other sequencingtechnologies may profit from electric field capture of nucleic acids atdiscrete locations on a flow cell or other sequencing device. Theattached Figures are meant to be considered as integral parts of thespecification and description of the invention. The following examplesare offered to illustrate, but not to limit the disclosure.

Preparation of Library Capture Surface of the Flow Cell

In the embodiment depicted in FIG. 1A, the displayed portion of a flowcell 100 includes two electrodes 102, 104 disposed on a solid support106 and an attachment layer 108. In this example, a silane-freeacrylamide (SFA) coating serves as the attachment layer 108.Amplification primers 110 are immobilized in the attachment layer 108.In this embodiment, paired-end (PE) primers serve as the amplificationprimers 110. The electrodes 102, 104 in the embodiment of FIG. 1A aremade of gold.

FIG. 1B presents a top view 120 and a side view 130 of a library capturesurface such as a lane of a flow cell such as cell 100 from FIG. 1A. Inboth views, six electrodes 103 are disposed in a row down the lane ofthe flow cell on support 106 (attachment layer not depicted). A topplate 107 defines an upper surface of the flow cell. The electrodes areconnected to electrical leads that can independently deliver a positiveor negative charge to each electrode. A flow cell may include any numberof lanes as can fit on the flow cell. In certain embodiments, a flowcell includes 6-12 lanes.

Library Preparation

In some embodiments, the DNA to be sequenced is fragmented to adesignated optimal length. Because DNA fragmentation does not result inhomogeneous, blunt-ended fragments, end repair may be performed toensure that each molecule is free of overhangs, and contains 5′phosphate and 3′ hydroxyl groups. Libraries containing blunt-ends can beused directly in an adaptor ligation step. For some libraries,incorporation of a non-templated deoxyadenosine 5′-monophosphate (dAMP)onto the 3′ end of blunted DNA fragments, a process known as dA-tailing,may be used to prevent concatamer formation during downstream ligationsteps, and enable DNA fragments to be ligated to adaptors withcomplementary dT-overhangs. The desired adaptor ligated DNA size may beselected via gel electrophoresis before amplification by the polymerasechain reaction (PCR).

In some embodiments, the library preparation step may be accomplishedoutside of the flow cell. In other embodiments, the library preparationstep may occur in the flow cell, using, e.g., transposon compositionsimmobilized to the solid support of the flow cell. In these embodiments,the transposon compositions may fragment and tag DNA fragments toprepare the library for seeding, clustering, amplification, andsequencing. More details regarding in-line sample preparation areprovided in a later section of this disclosure.

Hybridizing a First Polynucleotide Library

In this embodiment, a negative or neutral charge is applied to a firstelectrode and a positive charge is applied to a second electrode. Afirst library is then delivered to a library sequencing region such as alane flow cell. The library sequencing region contains the librarycapture surface with its associated plurality of electrodes. Becausepolynucleotides are negatively charged, the polynucleotides of the firstlibrary are attracted to the positively charged electrode and willpredominantly hybridize with amplification primers proximate to thepositive electrode. This localizes one library over one positivelycharged electrode or a set of positively charged electrodes.

FIG. 2A depicts a negative charge being applied to the first electrode202 and a positive charge being applied to the second electrode 204. Thepolynucleotides 206, 208, 210 of the first library are then drawn to thelibrary capture surface above the positive electrode 204 where theyhybridize with the amplification primers 212 proximate to the positiveelectrode 204. Note that capture moieties other than primers may beused. Such moieties may be nucleic acid strands or other species.

FIG. 2B presents a top view 220 and a side view 230 of a library capturesurface, such as a lane of a flow cell. FIG. 2B depicts the firstpolynucleotide library 222 being delivered down the lane of the flowcell. The side view 230 shows the repellant force 232 of the fiveelectrodes 234, 236, 238, 240, 242 that are negatively charged, and theattractive force 248 of the last electrode 244 that is positivelycharged and the polynucleotides hybridizing to the last electrode 244.The polynucleotides 246 of the first library predominantly hybridizewith the amplification primers proximate to the positively chargedelectrode 244.

In some implementations, the process applies a positive charge to two ormore electrodes when a first library passes through the flow cell (orportion thereof) where the two or more activated electrodes reside.Other electrodes in the flow cell (or portion thereof) would not beactivated.

FIG. 2C presents a top view 240 and a side view 250 of a library capturesurface, such as a lane of a flow cell. FIG. 2C depicts the last twoelectrodes 252, 254 being activated with a positive charge as the firstlibrary 256 is delivered down the lane of the flow cell. In thisembodiment, the polynucleotides of the first library 258 will hybridizepredominantly with amplification primers located in the general area ofthe last two electrodes 252, 254. The two concurrently activatedelectrodes need not be adjacent to one another.

Hybridizing a Second Polynucleotide Library

Returning to the example of FIG. 2A, after polynucleotide members 206,208, 210 of the first library are captured at a first location on thelibrary capture surface, a negative charge is applied to the secondelectrode 204 and a positive charge is applied to the first electrode202, and a second library is delivered through the flow cell. Thepolynucleotides of the second library will then predominantly hybridizewith primers proximate to the first electrode.

FIG. 3A depicts the left electrode 312 being positively charged whilethe second library 314 is delivered over the electrodes, where thepolynucleotides of the second library 314 then hybridize toamplification primers 316 near the left electrode 312. The result isthat the polynucleotides of the first 318 and second 314 libraries arespatially isolated within a lane of a flow cell.

FIG. 3B presents a top view 320 and a side view 330 of a library capturesurface, such as a lane of a flow cell. FIG. 3B depicts an example ofapplying a positive charge to a second electrode 322 while delivering asecond polynucleotide library 324 down the lane of the flow cell,accomplishing spatial isolation of two libraries 328, 326 within a laneof a flow cell.

FIG. 3C presents a top view 340 and a side view 350 of a library capturesurface, such as a lane of a flow cell. FIG. 3C depicts an example ofapplying a positive charge to a second set of electrodes 342 whiledelivering a second polynucleotide library 344 down the lane of the flowcell, both accomplishing spatial isolation of two libraries 346, 344within a lane of a flow cell.

In some embodiments, additional polynucleotide libraries may bedelivered through the lane of the flow cell while activating differentelectrodes with positive charges. For example, a third electrode or athird set of electrodes may be positively charged while delivering athird library through the flow cell. Additional libraries can bedelivered and selectively captured in the same manner.

Amplification and Sequencing

Once the two libraries have been hybridized in locations proximate tothe electrodes, the remaining operations of a standard protocol formultiplexed sequencing may be performed. For example, cluster generationand sequencing may be executed in a manner that concurrently sequencesall the captured libraries. Examples of these additional steps will beprovided below.

FIG. 3D shows a flow chart of a process of sequentially flowing one ormore libraries through a library sequencing region while activatingdifferent electrodes for multiplexed sequencing of the libraries.Initially, at block 301, one or more electrodes are selected andactivated from a plurality of electrodes proximate a library capturesurface by applying a positive potential. In some embodiments, anegative or neutral potential may be applied to the non-selectedelectrodes. At block 302, a selected library or libraries are flowed toa library sequencing region such that polynucleotides from the selectedlibrary or libraries are capture on the capture surface proximate theactivated electrodes. Because the polynucleotides have a negativecharge, they are attracted to the positively charged activatedelectrodes and will hybridize with the primers of the capture surfaceproximate the activated electrodes. At block 303, the unboundpolynucleotides are washed away from the library sequencing region,leaving behind the polynucleotides that are hybridized to the capturesurface primers proximate the library sequencing region. At block 304, adetermination is made as to whether any further libraries are to becaptured. If the answer is yes, then at block 305, a new library orlibraries is selected to be flowed through the library sequencingregion, and new electrodes are selected for activation. The process thenreturns to blocks 301, 302, and 303, where the new electrodes areactivated by applying a positive potential, the new libraries are flowedthrough the library sequencing region such that polynucleotides of thenew libraries are captured on the capture surface proximate theactivated electrode, and the unbound polynucleotides are washed away. Ifthe answer at block 304 is no, the process moves on to block 306, wherethe bound polynucleotides are amplified to form clusters proximate theelectrodes. In some embodiments, the amplification step is optional. Atblock 307, the bound polynucleotides or clusters undergo sequencing toidentify the nucleic acid sequences of the bound polynucleotides.

Applications

Some embodiments utilize separate electrodes to spatially isolate onelibrary from another in a flow cell lane for multiplexed sequencing.Spatial isolation allows libraries to be distinguished from one another,so that more distinct libraries may be sequenced at one time on a givenflow cell, ultimately reducing the sequencing time and cost for a DNAsample. Whereas current technology allows libraries to be distinguishedby the lane of a flow cell where a library is located, the disclosedembodiments allow, e.g., 6-12 libraries to be clustered and sequenced ina single flow cell lane without barcoding.

Conventional mechanisms for distinguishing libraries often utilizebarcoding, in which an index (or barcode) is attached to eachpolynucleotide of a library. The barcode is a unique nucleic acidsequence. Barcode identification requires that a sequencer read thebarcode, which may result in some loss of signal. Additionally, thelibrary preparation portion of the process must include steps that applythe same barcode to all members of a library. Some implementationsdisclosed herein reduce sequencing time by providing a method ofspatially indexing multiple libraries without requiring that a barcodebe attached to each polynucleotide of a library. This eliminates thetime required to read the polynucleotide barcodes. This also reduces thenumber of reagents required to prepare a sample for sequencing, sincethe barcodes do not need to be attached to the polynucleotide members ofthe library.

Certain embodiments use both barcoding and spatial separation toseparate libraries. In some embodiments, this permits high confidencethat libraries are segregated, which is relevant because regulatoryagencies typically require that diagnostic techniques contain rigoroussafeguards against cross contamination. Further, use of barcoding andspatial separation together may allow larger numbers of libraries to beprocessed together by multiplexing.

Another advantage of the claimed systems and methods is that the amountof time required to capture the polynucleotide libraries in the flowcell may be reduced. Using current technology such as that embodied inan Illumina HiSeq® sequencer, typically more than 15 minutes is requiredfor sufficient DNA from a library to be hybridized in the flow celllane. Some embodiments of current technology have demonstrated that15-50% of DNA is captured by the primers during hybridization over a 20minute period. In these tests, capture efficiency may be estimated usingobtained cluster density divided by input library concentration.

Utilizing electrodes in some of the disclosed embodiments, sufficienthybridization of the libraries to the primers proximate to theelectrodes may be accomplished in less than one minute. FIG. 4demonstrates that in some embodiments, about 40% of the transported DNAmay be captured by immobilized primers within one minute. This rapidhybridization time facilitates sequential introduction of multiplesamples into a flow cell lane. Additionally, since a greater percentageof the polynucleotides that are delivered through the flow cell arecaptured and hybridized when the claimed systems and methods are used, asmaller sample is required.

Moreover, current capture efficiency without electrodes is sufficientdue to high concentrations of amplification primers on the flow celllibrary capture surface. For example, current technology may employpaired end primers at a concentration of about 1000-5000 primers persquare micrometer. Applying an electric field with electrodes canincrease the speed of hybridization and/or enable the use of lowerconcentrations of target polynucleotides for flow cell capture-basedsequencing methods where the concentration of capture primers for targetpolynucleotides is significantly lower, e.g., more than 100 times lower,than that of the concentration of paired end primers in a conventionalflow cell.

Electrodes and their Application in Sequencing

Electrical Properties

Generally, the electrodes should be designed and placed to apply alocalized positive electrical field sufficiently great to pullpolynucleotide molecules out of a fluid and onto a region of a librarycapture surface selectively activated positive electrical field. Thematerial for the electrodes should allow reliable and efficientgeneration of such electric field. To accomplish this, the electrodesmay have a conductance in the approximate range of about 250 nS-1 μS. Insome embodiments, the electrodes are made of a high conductivitymaterial such as a metal or a conductive oxide, e.g., gold or indium tinoxide (ITO). In some embodiments, the electrodes are made of silver,tin, titanium, copper, platinum, palladium, polysilicon, or carbon. Theelectrodes may take any shape suitable for capturing and spatiallyisolating captured nucleic acids. For example, the electrodes may besubstantially round-shaped or polygonally shaped, e.g., rectangular. Insome embodiments, the electrodes are shaped as rectangles that span thewidth of a flow cell or a lane of a flow cell on which the electrode isdisposed. In such embodiments, two to twelve rectangular-shapedelectrodes are disposed in a line along the solid support of the flowcell, along the direction of fluid flow, e.g., perpendicular to thelanes of the flow cell.

Application of an electric potential to an electrode may becharacterized by various parameters. For example, the positive voltageapplied to an activated electrode may be in the approximate range ofabout 0.5-3V, depending on the electrode material and the polynucleotideacid carrier fluid used. In particular, the applied voltage may be belowthe voltage at which electrolysis of water occurs, and at or above thevoltage at which the redox reaction of a reducing agent additive occurs.The current produced during the application of the positive electricpotential to the electrode may be in the approximate range of about 250nA-5 uA. The current density produced may be in the approximate range ofabout 5-20 uA/mm². The electric field adjacent the electrode andamplification primers may be in the approximate range of about10-200V/cm.

In certain embodiments, rectangular electrodes have a width (directionperpendicular to the direction of flow) that spans most or all of thewidth of a lane of a flow cell. Further, the electrodes may have alength (direction of flow) that allows at least about five electrodesdisposed along the length of a flow cell lane. As an example, anelectrode may have a width of between about 1 um and 20 mm, and have alength of between about 1 um and 100 mm. The total surface area of anelectrode may be between about 100 um² and 200 mm². The separationbetween adjacent electrodes should be sufficient to prevent chargeapplied to one electrode from leaking to an adjacent electrode. Incertain embodiments, the minimum separation distance is between about 10micrometers and 1 millimeter, or between about 20 and 50 micrometers.

FIG. 5 illustrates that in some flow cell designs, the electric fieldgenerated by an electrode 510 is approximately constant across theheight of the flow cell lane from the bottom to the top of the flowcell. Further, examples of electrode configurations and dimensions aredescribed in the Examples section of the disclosure below.

Multiplexed Sequencing of Multiple Libraries

Spatial isolation of libraries onto different electrodes by an electricfield provides a new dimension to multiplexing. Using multiple flow celllanes introduces one dimension of multiplexing, as different librariesmay be loaded into different lanes to spatially separate the differentlibraries. A flow cell with eight lanes may be used to sequence eightdifferent libraries at one time.

Utilizing barcoding, in which an index (or barcode) is attached to eachpolynucleotide of a library, introduces another dimension ofmultiplexing. Therefore, if, within a single flow cell lane, eightdifferent libraries are introduced, each library having a differentbarcode attached to its polynucleotides, then the flow cell with eightlanes and barcoding may be used to sequence 64 different libraries atone time.

Introducing the spatial separation onto different electrodes of a flowcell lane by an electric field provides yet another dimension tomultiplexing. A flow cell 600 such as the one depicted in FIG. 6, whichhas eight flow cell lanes 610 and eight electrodes 620, and whichutilizes eight different barcodes, allows for sequentially loading eightsets of libraries into each flow cell lane, where each set of librariesincludes eight libraries that are distinguishable by their barcodes.Such a flow cell is able to sequence 512 different libraries at onetime.

In Line Sample Preparation

In some embodiments, the library preparation, wherein a DNA sample isfragmented to a designated length and adapters are ligated to the endsof the fragments, is performed outside of the flow cell. Certain methodscurrently used for fragmentation and tagging of double-stranded DNA foruse in next-generation sequencing may be wasteful of the DNA, requireexpensive instruments for fragmentation, and the procedures forfragmentation, tagging and recovering tagged DNA fragments aredifficult, tedious, laborious, time-consuming, inefficient, costly,require relatively large amounts of sample nucleic acids. In addition,many of these methods generate tagged DNA fragments that are not fullyrepresentative of the sequences contained in the sample nucleic acidsfrom which they were generated.

In some embodiments described herein, some procedures required forlibrary preparation are accomplished in the lane of the flow cell, asdescribed in U.S. patent application Ser. No. 13/790,220, which isincorporated by reference in its entirety herein. In these embodiments,transposon compositions immobilized to a solid support are used tofragment and tag the DNA.

In these embodiments, the method of preparing an immobilized library oftagged DNA fragments comprises: (a) providing a solid support havingtransposome complexes immobilized thereon, wherein the transposomecomplexes comprise a transposase bound to a first polynucleotide, thefirst polynucleotide comprising (i) a 3′ portion comprising a transposonend sequence, and (ii) a first tag comprising a first tag domain; and(b) applying a target DNA to the solid support under conditions wherebythe target DNA is fragmented by the transposome complexes, and the 3′transposon end sequence of the first polynucleotide is transferred to a5′ end of at least one strand of the fragments; thereby producing animmobilized library of double-stranded fragments wherein at least onestrand is 5′-tagged with the first tag. In some embodiments, thetransposome complexes comprise a second polynucleotide comprising aregion complementary to said transposon end sequence. The methods canfurther comprise (c) providing transposome complexes in solution andcontacting the transposome complexes with the immobilized fragmentsunder conditions whereby the target DNA is fragmented by the transposomecomplexes in solution; thereby obtaining immobilized nucleic acidfragments having one end in solution. In some embodiments, thetransposome complexes in solution can comprise a second tag, such thatthe method generates immobilized nucleic acid fragments having a secondtag, the second tag in solution. The first and second tags can bedifferent or the same.

In some embodiments, the solid support may include a library of taggedDNA fragments immobilized thereon. For example, the solid supports mayhave transposome complexes immobilized thereon, wherein the transposomecomplexes comprise a transposase bound to a first polynucleotide, thepolynucleotide comprising (i) a 3′ portion comprising a transposon endsequence, and (ii) a first tag comprising a first tag domain.

In some embodiments, a flow cell may be generated by immobilizing aplurality of transposome complexes to a solid support, the transposomecomplexes comprising a transposase bound to a first polynucleotide, thefirst polynucleotide comprising (i) a 3′ portion comprising a transposonend sequence, and (ii) a first tag comprising a first tag domain.

As depicted in FIG. 7, an electric field generated by one or moreelectrodes 710 in a flow cell may facilitate inline sample preparationusing immobilized Tn5 transposons 720. For example, a first genomic DNAsample 722 may be introduced into the flow cell while one electrode in atarget area is at a positive or zero potential and while the rest of theelectrodes outside the target area are at a negative potential. Theimmobilized Tn5 transposons will only incorporate adaptors onto genomicDNA in the target area. In FIG. 7, the upper panels 740, 742 present topviews of two lanes of a flow cell and the lower panels 750, 752 presentside views of the same flow cell cut through the lanes in having apositively charged electrode. Transposons 720 are shown as immobilizedegg-shaped proteins and sample DNA 722 is shown as string-likestructures. Positively-charged transposon inhibitors 724, which areshown as black dots in FIG. 7, are attracted to negative electrodes toinhibit adaptor incorporation outside of the target region 710. The leftpanels of FIG. 7 illustrate the flow cell lanes when a first set ofpolynucleotides 722 is introduced and the right panels illustrate theflow cell lanes when a second set of polynucleotides 732 is introduced.The next set may be introduced with activation of a third electrode orset of electrodes, and so forth. The polynucleotides in a set may belongto a library, a sample, etc. In other embodiments, a positively chargedTn5 inhibitor 724 can be simultaneously delivered and attracted tonegatively charged electrodes to inhibit adaptor incorporation outsideof the target area.

Fluid Medium Provided to Library Sequencing Region

The polynucleotides of a library are delivered to the library sequencingregion of a flow cell or other sequencing device via a carrier fluid.The polynucleotides are carried along with fluid and may randomly attachto the capture surface along the way. However, most of thepolynucleotides are captured at a region (or regions) of the capturesurface where one or more electrodes are activated with a positivecharge. The positive electric field exerts an attractive electrostaticforce on the polynucleotides and pulls them out of the carrier fluid andonto the capture surface.

The carrier fluid may contain water and various additives to buffer andotherwise stabilize the polynucleotides of the library. In someembodiments, the buffer used for electric field directed hybridizationmay be a relatively low conductivity buffer with a reducing agent, andhaving a pH in the range of about 6-9. One approach to designing acarrier fluid for electric field hybridization uses a buffer suitablefor DNA electrophoresis (such as one containing Tris, Bis-Tris, orimidazole—e.g., 1×TBE) to which is added a reducing agent. Theelectrophoresis buffer provides suitable conductivity and pH such thatDNA is negatively charged. The reaction of reducing agent, when exposedto an electric field from the electrodes, provides current that candrive DNA movement without inducing electrolysis of water. In oneembodiment, the carrier fluid contains 1×TBE (89Mm Tris, 89 mM Boricacid, 2 mM EDTA, pH=8.3).

In some embodiments, protective chemistry is utilized in the buffersolution to prevent damage to the primers of the capture surface and tothe polynucleotides in the library. In the absence of a protectivereducing agent in the buffer solution, application of an electricalcurrent to the buffer may, at a sufficient voltage, cause someelectrolysis of water molecules, producing molecular oxygen, which maydamage the nucleic acid amplification primers and/or the polynucleotidelibrary. Damage to the amplification primers may cause them to lose thecapability to bind to the complementary sequences of the librarypolynucleotides. FIG. 8 demonstrates how incrementing the voltageincreases the electrical current produced and the point at whichelectrolysis begins to occur. In theory, the library processing systemcould include a voltage controller to prevent the sequencing regionvoltage from exceeding a threshold after which electrolysis occurs. Asillustrated in FIG. 8, however, the transition 810 between electrolysisand no electrolysis can be very abrupt.

Adding to the buffer a protective reducing agent, which undergoeselectrolysis at a potential lower than the potential at which waterundergoes electrolysis, permits the electric field to be generated forcapture/hybridization of the polynucleotides without generatingmolecular oxygen. Including an appropriate reducing agent, such asbeta-mercaptoethanol, produces a protective redox reaction that keepsthe local potential in the sequencing region below the potential atwhich oxygen forms. The process generates sufficient potential to inducemobility of polynucleotides toward the capture surface but withsubstantially reduced generation of molecular oxygen. Other reducingagents that may be used include: dithiothreitol, alpha-thiolglycerol,hydroquinone, or any electroactive species with an oxidation potentiallower than that of water on the electrodes used in the disclosedembodiments.

In many implementations, the buffer employs a relatively highconcentration of reducing agent to produce sufficient current; however,the upper concentration limit may be limited based on (a) solubility ofthe reducing agent and (b) addition of the reducing agent cannotinducing significant change (<1 pH unit) to the buffer pH. For2-mercaptoethanol, an example of a suitable range is about 0.7-2.8M; forHydroquinone an example of a suitable range is about be 100-500 mM.

In one example, a reducing agent used in combination with 1×TBE bufferon Gold electrodes is 1.4M 2-mercaptoethanol and 10 mM Dithiothreitol(although just 1.4M 2-mercaptoethanol may be sufficient). In anotherexample, a reducing agent used in combination with 1×TBE buffer on ITOelectrode is 250 mM hydroquinone.

In some embodiments, two or more reducing agents may be used together inthe buffer. Generally, in some embodiments, the potential applied toattract the polynucleotides toward the capture surface is above thepotential at which the reducing agent undergoes a redox reaction, butbelow the potential at which electrolysis of water occurs on thespecific electrode material. The redox reaction of the protective agentmoderates the actual potential experienced within the sequencing region,preventing that potential from reaching a level at which waterelectrolysis may occur. Further, the products of the protective redoxreaction should be benign to the library polynucleotides and anyoligonucleotides on the capture surface. An example of a benignbyproduct is a disulfide produced by oxidation of a mercaptan.

FIG. 9 demonstrates the protective effect of adding a reducing agent,such as 2-mercaptoethanol (beta-mercaptoethanol), to the buffer. Thecircles 910, 920 indicate where electrodes are exposed to solution andwhere there is electric field. Voltage application in a histidine buffersolution without reducing agent leads to electrolysis, which damages theamplification primers and negatively impacts their ability to bind tothe library polynucleotides and support bridge amplification. Voltageapplication in a 1×TBE/2-mercaptoethanol buffer generates currentthrough the oxidation of 2-mercaptoethanol, which is less damaging toprimers; hence, the primers can support downstream cluster generation.

Cross-Contamination

Hybridization using an electric field may result in somecross-contamination if there is insufficient repulsion from the negativeelectrodes such that a polynucleotide hybridizes with a capture primernear a negative electrode. Another potential source ofcross-contamination is when a hybridized polynucleotide, whichhybridized with a primer when the nearby electrode had a positivecharge, is removed from the electrode when a negative charge issubsequently applied to the nearby electrode.

FIG. 10A presents a flow chart in which these sources of contaminationare addressed by controlling flow to a flow cell or other sequencingregion and by controlling potential to multiple electrodes in the flowcell. Initially, in block 1010, a solution containing a seed libraryflows over an electrode while the electrode has a potential of, e.g.,+0.95V. Thereafter, at block 1012, the sequencing region, including theelectrodes, is washed to remove unbound polynucleotides from the seedlibrary. Next, at block 1014, the controller applies a negative bias(e.g., −0.95 V) to the electrode, while applying a positive bias toanother electrode and flowing a different library over the electrodes.Thereafter, at block 1016, the sequencing region is again washed toremove unbound polynucleotides. The process of applying a positivevoltage to another electrode and a negative voltage to the remainingelectrodes followed by washing may be repeated for one or moreadditional libraries. After all libraries are captured, the systemsequences them simultaneously. In the embodiment of FIG. 10A, this isaccomplished by bridge amplification at block 1018, followed bylinearization and sequencing at block 1020.

FIG. 10B shows an image of channel T following the first base read (T)after the flow described in FIG. 10A is executed. The figure shows thatapplying a 0.95V charge to the target electrodes while delivering thepolynucleotide library through a flow cell, followed by reversing thecharge resulted in only 0.7% rate of cross-contamination onto thenon-target electrodes in this example. Further improvement can beachieved by refining the capture and washing operations.

Capture Conditions for Different Electrode Materials

As explained, gold electrodes may be used to produce the electric fieldto manipulate the polynucleotide libraries being delivered through theflow cell. In other embodiments, ITO electrodes on glass slides may beused. Whereas a voltage of approximately 0.9-1V is sufficient to attractthe polynucleotides to the capture primers near the charged electrodewhen the electrode is made of gold, FIGS. 11A and 11B illustrate that avoltage of approximately 3V may concentrate DNA on ITO electrodes 1110in a buffer without reducing agent. FIG. 12A demonstrates that theaddition of hydroquinone to the buffer enables DNA concentration at avoltage of less than 1V using ITO electrodes 1210. FIGS. 12B and 12Cdemonstrate that oxidation of hydroquinone occurs at a voltage lowerthan 1V (well below the potential at which water electrolyzes), whichmeans that hydroquinone is a suitable reducing agent for the buffer whenITO electrodes are used.

Bridge Amplification

Once the library polynucleotides have been selectively captured asdescribed above, they may be sequenced by an appropriate technique. Insome embodiments, the captured polynucleotides are amplified to formclusters which are then sequenced by synthesis. This is not the onlysequencing technique suitable for use with this disclosure, but it willbe explained as an example in this and the following section.

The captured target polynucleotides hybridize with the amplificationprimers on the library capture surface, captured the capturedpolynucleotides can be amplified. Optionally, the amplificationcomprises using the primers on the support. Alternatively, theamplification can comprise using one primer in solution and one primeron the support. In some embodiments, amplification produces clusters ofamplified target nucleic acid molecules. Generally amplificationreactions use at least two amplification oligonucleotides, often denoted‘forward’ and ‘reverse’ primers. Generally amplificationoligonucleotides are single stranded polynucleotide structures. They mayalso contain a mixture of natural or non-natural bases and also naturaland non-natural backbone linkages, provided, at least in someembodiments, that any non-natural modifications do not permanently orirreversibly preclude function as a primer—that being defined as theability to anneal to a template polynucleotide strand during conditionsof an extension or amplification reaction and to act as an initiationpoint for the synthesis of a new polynucleotide strand complementary tothe annealed template strand. Primers may additionally comprisenon-nucleotide chemical modifications, for example to facilitatecovalent attachment of the primer to a support. Certain chemicalmodifications may themselves improve the function of the molecule as aprimer or may provide some other useful functionality, such as providinga cleavage site that enables the primer (or an extended polynucleotidestrand derived therefrom) to be cleaved from a support.

Nucleic acid amplification includes the process of amplifying orincreasing the numbers of a nucleic acid template and/or of a complementthereof that are present, by producing one or more copies of thetemplate and/or or its complement. Amplification can be carried out by avariety of known methods under conditions including, but not limited to,thermocycling amplification or isothermal amplification. For example,methods for carrying out amplification are described in U.S. PublicationNo. 2009/0226975; WO 98/44151; WO 00/18957; WO 02/46456; WO 06/064199;and WO 07/010251; which are incorporated by reference herein in theirentireties. Thus, amplification can occur on the surface to which thenucleic acid molecules are attached. This type of amplification can bereferred to as solid phase amplification, which when used in referenceto nucleic acids, refers to any nucleic acid amplification reactioncarried out on or in association with a surface (e.g., a support). Forexample, all or a portion of the amplified products are synthesized byextension of an immobilized primer. Solid phase amplification reactionsare analogous to standard solution phase amplifications except that atleast one of the amplification oligonucleotides is immobilized on asurface (e.g., a solid support).

Solid-phase amplification may comprise a nucleic acid amplificationreaction comprising only one species of oligonucleotide primerimmobilized to a surface. Alternatively, the surface may comprise aplurality of first and second different immobilized oligonucleotideprimer species. Solid-phase amplification may comprise a nucleic acidamplification reaction comprising one species of oligonucleotide primerimmobilized on a solid surface and a second different oligonucleotideprimer species in solution. Solid phase nucleic acid amplificationreactions generally comprise at least one of two different types ofnucleic acid amplification, interfacial and surface (or bridge)amplification. For instance, in interfacial amplification, the solidsupport comprises a template nucleic acid molecule that is indirectlyimmobilized to the solid support by hybridization to an immobilizedoligonucleotide primer, the immobilized primer may be extended in thecourse of a polymerase-catalyzed, template-directed elongation reaction(e.g., primer extension) to generate an immobilized polynucleotidemolecule that remains attached to the solid support. After the extensionphase, the nucleic acids (e.g., template and its complementary product)are denatured such that the template nucleic acid molecule is releasedinto solution and made available for hybridization to anotherimmobilized oligonucleotide primer. The template nucleic acid moleculemay be made available in 1, 2, 3, 4, 5 or more rounds of primerextension or may be washed out of the reaction after 1, 2, 3, 4, 5 ormore rounds of primer extension.

In surface (or bridge) amplification, an immobilized nucleic acidmolecule hybridizes to an immobilized oligonucleotide primer. The 3′ endof the immobilized nucleic acid molecule provides the template for apolymerase-catalyzed, template-directed elongation reaction (e.g.,primer extension) extending from the immobilized oligonucleotide primer.The resulting double-stranded product “bridges” the two primers and bothstrands are covalently attached to the support. In the next cycle,following denaturation that yields a pair of single strands (theimmobilized template and the extended-primer product) immobilized to thesolid support, both immobilized strands can serve as templates for newprimer extension.

Optionally, amplification of the adapter-target-adapters or library ofnucleic acid sequences results in clustered arrays of nucleic acidcolonies, analogous to those described in U.S. Pat. No. 7,115,400; U.S.Publication No. 2005/01 00900; WO 00/18957; and WO 98/44151, which areincorporated by reference herein in their entireties. Clusters andcolonies are used interchangeably and refer to a plurality of copies ofa nucleic acid sequence and/or complements thereof attached to asurface. Typically, the cluster comprises a plurality of copies of anucleic acid sequence and/or complements thereof, attached via their 5′termini to the surface. The copies of nucleic acid sequences making upthe clusters may be in a single or double stranded form.

Clusters may be detected, for example, using a suitable imaging means,such as, a confocal imaging device or a charge coupled device (CCD)camera. Exemplary imaging devices include, but are not limited to, thosedescribed in U.S. Pat. Nos. 7,329,860; 5,754,291; and 5,981,956; and WO2007/123744, each of which is herein incorporated by reference in itsentirety. The imaging means may be used to determine a referenceposition in a cluster or in a plurality of clusters on the surface, suchas the location, boundary, diameter, area, shape, overlap and/or centerof one or a plurality of clusters (and/or of a detectable signaloriginating therefrom). Such a reference position may be recorded,documented, annotated, converted into an interpretable signal, or thelike, to yield meaningful information. The signal may, for instance,take the form of a detectable optical signal emanating from a definedand identifiable location, such as a fluorescent signal, or may be adetectable signal originating from any other detectable label asprovided herein. The reference position of a signal generated from twoor more clusters may be used to determine the actual physical positionon the surface of two clusters that are related by way of being thesites for simultaneous sequence reads from different portions of acommon target nucleic acid.

Sequencing by Synthesis

Following amplification, the amplified target extension products ortarget nucleic acids can be sequenced. Optionally, the sequencingincludes sequencing-by-synthesis or sequencing-by-ligation.

Sequencing by synthesis, for example, is a technique wherein nucleotidesare added successively to a free 3′ hydroxyl group, typically providedby annealing of an oligonucleotide primer (e.g., a sequencing primer),resulting in synthesis of a nucleic acid chain in the 5′ to 3′direction. These and other sequencing reactions may be conducted on theherein described surfaces bearing nucleic acid clusters. The reactionscomprise one or a plurality of sequencing steps, each step comprisingdetermining the nucleotide incorporated into a nucleic acid chain andidentifying the position of the incorporated nucleotide on the surface.The nucleotides incorporated into the nucleic acid chain may bedescribed as sequencing nucleotides and may comprise one or moredetectable labels. Suitable detectable labels, include, but are notlimited to, haptens, radionucleotides, enzymes, fluorescent labels,chemiluminescent labels, and/or chromogenic agents. One method fordetecting fluorescently labeled nucleotides comprises using laser lightof a wavelength specific for the labeled nucleotides, or the use ofother suitable sources of illumination. The fluorescence from the labelon the nucleotide may be detected by a CCD camera or other suitabledetection means. Suitable instrumentation for recording images ofclustered arrays is described in WO 07/123744, the contents of which areincorporated herein by reference herein in its entirety.

Various additional aspects regarding sequencing by synthesis proceduresand methods that can be utilized with the systems and devices herein aredescribed in, e.g., WO04018497, WO04018493 and U.S. Pat. No. 7,057,026(nucleotides), WO05024010 and WO06120433 (polymerases), WO05065814(surface attachment techniques), and WO 9844151, WO06064199 andWO07010251, the contents of each of which are incorporated herein byreference in their entirety.

Optionally, cycle sequencing is accomplished by stepwise addition ofreversible terminator nucleotides containing, for example, a cleavableor photobleachable dye label as described, for example, in U.S. Pat.Nos. 7,427,673; 7,414,116; WO 04/018497; WO 91/06678; WO 071123744; andU.S. Pat. No. 7,057,026, the disclosures of which are incorporatedherein by reference in their entireties. The availability offluorescently labeled terminators in which both the termination can bereversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

Alternatively, pyrosequencing techniques may be employed. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi et al.,(1996) “Real-time DNA sequencing using detection of pyrophosphaterelease.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001)“Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11;Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method basedon real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos.6,210,891; 6,258,568; and 6,274,320, the disclosures of which areincorporated herein by reference in their entireties). Inpyrosequencing, released PPi can be detected by being immediatelyconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated is detected via luciferase-produced photons.

Additional exemplary sequencing-by-synthesis methods that can be usedwith the methods described herein include those described in U.S. PatentPublication Nos. 2007/0166705; 2006/0188901; 2006/0240439; 2006/0281109;2005/0100900; U.S. Pat. No. 7,057,026; WO 05/065814; WO 06/064199; WO07/010251, the disclosures of which are incorporated herein by referencein their entireties.

Alternatively, sequencing by ligation techniques are used. Suchtechniques use DNA ligase to incorporate oligonucleotides and identifythe incorporation of such oligonucleotides and are described in U.S.Pat. Nos. 6,969,488; 6,172,218; and 6,306,597; the disclosures of whichare incorporated herein by reference in their entireties. Other suitablealternative techniques include, for example, fluorescent in situsequencing (FISSEQ), and Massively Parallel Signature Sequencing (MPSS).

Apparatus Flow Cell Design

Various library capture devices may be provided with electrodes asdescribed herein. Flow cells can serve as such devices. In variousembodiments, the devices herein include one or more substrates uponwhich the nucleic acids to be sequenced are bound, attached orassociated. See, e.g., WO 9844151 or WO0246456. In certain embodiments,a library sequencing region is within a channel or other area as part ofa “flow cell.” The flow cells used in the various embodiments caninclude millions of individual nucleic acid clusters, e.g., about 2-8million clusters per channel. Each of such clusters can give readlengths of at least 25 bases for DNA sequencing and 20 bases for geneexpression analysis. In certain embodiments, the flow cells herein cangenerate a gigabase (one billion bases) of sequence per run (e.g., 5million nucleic acid clusters per channel, 8 channels per flow cell, 25bases per polynucleotide).

FIGS. 13A and 13B display one exemplary embodiment of a flow cell. Ascan be seen, the particular flow cell embodiment, flow cell 1300,includes a base layer 1310 (e.g., of borosilicate glass 1000 um indepth), channel layer 1320 (e.g., of etched silicon 100 um in depth)overlaid upon the base layer, and cover, or top, layer 1330 (e.g., 300um in depth). When the layers are assembled together, enclosed channelsare formed having inlet/outlets at either end through the cover. As willbe apparent from the description of additional embodiments below, someflow cells include openings for the channels on the bottom.

The channeled layer can optionally be constructed using standardphotolithographic methods, with which those of skill in the art will befamiliar. One such method, which can be used in some embodiments,involves exposing a 100 um layer of silicon and etching away the exposedchannel using Deep Reactive Ion Etching or wet etching.

It will be appreciated that while particular flow cell configurationsare presented herein, such configurations should not be taken aslimiting. Thus, for example, various flow cells herein can includedifferent numbers of channels (e.g., 1 channel, 2 or more channels, 4 ormore channels, or 6, 8, 10, 16 or more channels, etc. Additionally,various flow cells can include channels of different depths and/orwidths (different both between channels in different flow cells anddifferent between channels within the same flow cell). For example,while the channels formed in the cell in FIG. 13B are 100 um deep, otherembodiments can optionally comprise channels of greater depth (e.g., 500um) or lesser depth (e.g., 50 um). Additional exemplary flow celldesigns are shown in FIGS. 13C and 13D (e.g., a flow cell with “wide”channels, such as channels 1340 in FIG. 13C, having two channels with 8inlet and outlet ports (ports 1345—8 inlet and 8 outlet) to maintainflow uniformity and a center wall, such as wall 1350, for addedstructural support; or a flow cell with offset channels, such as the 16offset channels (channels 1380), etc.). The flow cells can be designedto maximize the collection of fluorescence from the illuminated surfaceand obtain diffraction limited imaging. For example, in the design shownin FIG. 13C, in particular embodiments, the light comes into the channelthrough 1000 um thick bottom layer 1360, which can be made ofborosilicate glass, fused silica or other material as described herein,and the emitted light travels through 100 um depth of aqueous solutionwithin the channel and 300 um depth of “top” layer material 1370.However, in some embodiments, the thickness of the “top” layer may beless than 300 um to prevent spherical aberrations and to image adiffraction limited spot. For example the thickness of the top layer canbe around 170 um for use with a standard diffraction limited opticalsystem. To use the thicker top layer without suffering from sphericalaberrations, the objective can optionally be custom designed, e.g., asdescribed herein.

In the various embodiments herein, the flow cells can be createdfrom/with a number of possible materials. For example, in someembodiments, the flow cells can comprise photosensitive glass(es) suchas Foturan® (Mikroglas, Mainz, Germany) or Fotoform® (Hoya, Tokyo,Japan) that can be formed and manipulated as necessary. Other possiblematerials can include plastics such as cyclic olefin copolymers (e.g.,Topas® (Ticona, Florence, Ky.) or Zeonor® (Zeon Chemicals, Louisville,Ky.)) which have excellent optical properties and can withstand elevatedtemperatures if need be (e.g., up to 100° C.). As will be apparent fromFIG. 4, the flow cells can comprise a number of different materialswithin the same cell. Thus, in some embodiments, the base layer, thewalls of the channels, and the top/cover layer can optionally be ofdifferent materials.

While the example in FIG. 13B shows a flow cell containing 3 layers,other embodiments can include 2 layers, e.g., a base layer havingchannels etched/ablated/formed within it and a top cover layer, etc.Additionally, other embodiments provide flow cells having only one layerwhich contains the flow channel etched/ablated/otherwise formed withinit.

In some embodiments, the flow cells are constructed from Foturan®.Foturan is a photosensitive glass which can be structured for a varietyof purposes. It combines various desired glass properties (e.g.,transparency, hardness, chemical and thermal resistance, etc.) and theability to achieve very fine structures with tight tolerances and highaspect ratios (hole depth/hole width). With Foturan® the smalleststructures possible are usually, e.g., 25 um with a roughness of 1 um.

FIG. 6 depicts how the flow cell design may be modified to include ITOelectrodes on the bottom plate 620, and an ITO coated top plate 630. Thebottom plate 620 may be patterned to include segments of ITO electrodes,so that a given lane of a flow cell may include 6-12 electrodes, eachseparately activatable. During capture, the top plate 630 may be held ata single potential while the lower electrodes are held at differentpotentials. For example, when a bottom electrode is held at about +0.6V,the top plate is held at about −0.3V to repel polynucleotides, pushingthem toward the bottom plate 620 and the positively chargedelectrode(s).

In some embodiments, the electrodes of the flow cell may be arrangedparallel to one another and perpendicular to the lanes of the flow cell.Some embodiments of the flow cell may include six to twelve rectangularelectrodes disposed in parallel along the flow cell. Electrodes may beembedded in flow cells having various dimensions, including dimensionsused in current Illumina® GA®, MiSeq®, HiSeq® platforms and others. Thedimensions of the flow cell may vary as long as the flow cell cansuccessfully interface with existing and future sequencing platforms.

FIG. 14 is an exploded view of a flow cell having eight lanes and eightelectrodes disposed along the bottom plate of the flow cell. FIG. 14depicts this configuration for electrodes in a flow cell. In thisembodiment, eight electrodes are placed in parallel along the bottomplate 1410 of the flow cell, and the electrodes 1420 run perpendicularto the lanes of the flow cell. This effectively partitions each lane ofthe flow cell into eight sections, allowing for eight differentlibraries to be sequenced on a single lane of the flow cell. In a flowcell having eight lanes, the flow cell may be used to sequence up to 64different polynucleotide libraries.

FIGS. 15A and 15B depict alternative electrode designs for a flow cell.FIG. 15A shows a planar view of two possible configurations ofelectrodes on a flow cell. FIG. 15B shows a side view of two possibleconfigurations of electrodes on a flow cell. As FIG. 15B demonstrates,the upper plate 1500 may be patterned with ITO either as a singleelectrode 1510 across the entire upper plate 1500, or as counterelectrodes 1520 that match the electrodes 1530 on the bottom plate 1502.When one of the bottom plate electrodes is positively charged, the upperplate 1500 may be held at a negative voltage to further pushpolynucleotides away from the top plate 1500 toward the amplificationprimers on the bottom plate 1502. In other embodiments, the counterelectrodes may reside outside of the flow cell surface, for example, atthe inlet and outlets of the flow cell lanes or on the outside surfaceof the upper plate.

Fabrication of Flow Cells with Electrodes

In some embodiments, electrodes can be fabricated on top and bottom flowcell surfaces using standard micro-fabrication techniques prior to flowcell assembly. For example, gold electrodes can be patterned by lift-offphotolithography; and ITO electrodes can be patterned byphotolithography and wet etching or laser ablation.

FIGS. 16A and 16B show an example of a design where the gold electrodesare partially covered and passivated by silicon nitride, so that theactive region for DNA manipulation is concentrated in the areas wherethe gold electrode is exposed. Silicon nitride, or any similar compoundmay be deposited on the flow cell to create areas of active DNAmanipulation and areas where the electrodes are not exposed. FIG. 16Ashows a planar view of such a design, in which the white circlesrepresent exposed gold electrodes, and the black circles 1620 representelectrodes that are passivated with nitrides. In one implementation, theexposed gold electrodes 1610 may be 20 um in diameter and spaced apartfrom each other by 10 um. FIG. 16B shows a side view of a flow cellcontaining two gold electrodes 1630, 1632 and deposits of siliconnitride 1634.

A polymer attachment layer anchors the amplification primers or othercapture moieties to the library capture surface of a flow cell. Apolymer attachment layer composed of silane-free acrylamide (SFA) may bedirectly deposited on Au and ITO electrodes without any chemicalmodifications of the electrodes. A polymer attachment comparable topoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) may beattached to silanized ITO electrodes.

One type of polymer attachment layer employs a hydrogel. In preparinghydrogel-based solid-supported molecular arrays, a hydrogel is formedand molecules displayed from it. These two features-formation of thehydrogel and construction of the array—may be effected sequentially orsimultaneously. Where the hydrogel is formed prior to formation of thearray, it is typically produced by allowing a mixture of co-monomers topolymerize. Generally, the mixture of co-monomers contain acrylamide andone or more co-monomers, the latter of which permit, in part, subsequentimmobilization of molecules of interest so as to form the moleculararray.

The co-monomers used to create the hydrogel typically contain afunctionality that serves to participate in crosslinking of the hydrogeland/or immobilize the hydrogel to the solid support and facilitateassociation with the target molecules of interest.

Clustered arrays may be formed on such solid-supported hydrogels bysolid phase nucleic acid amplification using forward and reverseamplification primers attached to the hydrogel at their 5′ ends, leadingto the production of clustered arrays of amplification products havingthe “bridged” structure. In order to maximize the efficiency ofsequencing reactions using templates derived from such bridged productsthere is a need for linearization methods which are compatible with thehydrogel surface and with subsequent nucleic acid sequencing reactions.

WO 00/31148 discloses polyacrylamide hydrogels and polyacrylamidehydrogel-based arrays in which a so-called polyacrylamide prepolymer isformed, optionally from acrylamide and an acrylic acid or an acrylicacid derivative containing a vinyl group. Crosslinking of the prepolymermay then be effected. The hydrogels so produced are solid-supported,preferably on glass. Functionalization of the solid-supported hydrogelmay also be effected.

WO 01/01143 describes technology similar to WO 00/31148 but differing inthat the hydrogel bears functionality capable of participating in a[2+2] photocycloaddition reaction with a biomolecule so as to formimmobilized arrays of such biomolecules. Dimethylmaleimide (DMI) is aparticularly preferred functionality. The use of [2+2]photocycloaddition reactions, in the context of polyacrylamide-basedmicroarray technology is also described in WO02/12566 and WO03/014392.

U.S. Pat. No. 6,465,178 discloses the use of reagent compositions inproviding activated slides for use in preparing microarrays of nucleicacids; the reagent compositions include acrylamide copolymers. Theactivated slides are stated to be particularly well suited to replaceconventional (e.g. silylated) glass slides in the preparation ofmicroarrays.

WO00/53812 discloses the preparation of polyacrylamide-based hydrogelarrays of DNA and the use of these arrays in replica amplification.

The solid upon which the hydrogel is supported is not limited to aparticular matrix or substrate. Indeed, this is one of the advantages ofthese embodiments: the same chemistry used to modify silica-basedsubstrates can be applied to other solid supports and allows the solidsupport to be adapted to suit any particular application to which it isdesired to be put rather than being constrained by the surface chemistryit is possible to perform on any given support. Solids which may be ofuse in the practice of the disclosed embodiments thus includesilica-based substrates, such as glass, fused silica and othersilica-containing materials; they may also be silicone hydrides orplastic materials such as polyethylene, polystyrene, poly(vinylchloride), polypropylene, nylons, polyesters, polycarbonates andpoly(methyl methacrylate). Example plastics material are poly(methylmethacrylate), polystyrene and cyclic olefin polymer substrates.Alternatively, other solid supports may be used such as gold, titaniumdioxide, or silicon supports. The foregoing lists are intended to beillustrative of, but not limited to, the disclosed embodiments. Incertain embodiments, the support is a silica-based material or plasticmaterial such as discussed herein.

Plastics-based substrates for molecular arrays may be relativelyinexpensive: the preparation of appropriate plastics-based substratesby, for example injection-molding, is generally cheaper than thepreparation, e.g. by etching and bonding, of silica-based substrates.Another advantage is the nearly limitless variety of plastics allowingfine-tuning of the optical properties of the support to suit theapplication for which it is intended or to which it may be put.

In certain embodiments, the support is silica-based but the shape of thesupport employed may be varied in accordance with the application forwhich the disclosed embodiments are practiced. Generally, however,slides of support material, such as silica, e.g. fused silica, are ofparticular utility in the preparation and subsequent integration ofmolecules. Of particular use in the practice of the disclosedembodiments are fused silica slides sold under the trade nameSPECTRASIL™. This notwithstanding, it will be evident to the skilledperson that the disclosed embodiments are equally applicable to otherpresentations of solid support (including silica-based supports), suchas beads, rods and the like.

Sequencing Systems

While total internal reflection microscopy has been used to image bothsingle and amplified molecules of DNA on surfaces, a robust, reliable,four color DNA sequencing platform (e.g., comprising heating systems,fluidic controls, uniform illumination, control of the optical beamshape, an autofocus system, and full software control of all components)is described herein.

The disclosed systems and devices may be used to analyze a large numberof different nucleic acid sequences from, e.g., clonally amplifiedsingle-molecule DNA arrays in flow cells, or from an array ofimmobilized beads. In particular, the systems and devices utilizeelectrode arrays configured to capture DNA libraries when a positivebias is applied, isolating DNA from different samples to differentregions of a flow cell for multiplexed sequencing. The systems hereinare optionally useful in, e.g., sequencing for comparative genomics(such as for genotyping, SNP discovery, BAC-end sequencing, chromosomebreakpoint mapping, and whole genome sequence assembly), tracking geneexpression, micro RNA sequence analysis, epigenomics (e.g., withmethylation mapping DNAsel hypersensitive site mapping or chromatinimmunoprecipitation), and aptamer and phage display librarycharacterization. Of course, those of skill in the art will readilyappreciate that the disclosed embodiments are also amenable to use formyriad other sequencing applications.

An illustrative embodiment is outlined in FIG. 17, which shows anexemplary TIRF imaging configuration of a backlight design embodiment.As can be seen in FIG. 17, fluid delivery module or device 1700 directsthe flow of reagents (e.g., fluorescent nucleotides, buffers, enzymes,cleavage reagents, etc.) to (and through) flow cell 1710 and waste valve1720. In particular embodiments, the flow cell comprises clusters ofnucleic acid sequences (e.g., of about 200-1000 bases in length) to besequenced which are optionally attached to the substrate of the flowcell, as well as optionally other components. The flow cell can alsocomprise an array of beads, where each bead optionally contains multiplecopies of a single sequence. The preparation of such beads can beperformed according to a variety of techniques, for example as describedin U.S. Pat. No. 6,172,218 or WO04069849 (Bead emulsion nucleic acidamplification).

The system also comprises temperature station actuator 1730 andheater/cooler 1735, which can optionally regulate the temperature ofconditions of the fluids within the flow cell. As explained below,various embodiments can comprise different configurations of theheating/cooling components. The flow cell is monitored, and sequencingis tracked, by camera system 1740 (e.g., a CCD camera) which caninteract with various filters within filter switching assembly 1745,lens objective 1742, and focusing laser/focusing laser assembly 1750.Laser device 1760 (e.g., an excitation laser within an assemblyoptionally comprising multiple lasers) acts to illuminate fluorescentsequencing reactions within the flow cell via laser illumination throughfiber optic 1761 (which can optionally comprise one or more re-imaginglenses, a fiber optic mounting, etc. Low watt lamp 1765, mirror 1780 andreverse dichroic 1785 are also presented in the embodiment shown. Seebelow. Additionally, mounting stage 1770, allows for proper alignmentand movement of the flow cell, temperature actuator, camera, etc. inrelation to the various components of the disclosed embodiments. Focus(z-axis) component 1775 can also aid in manipulation and positioning ofvarious components (e.g., a lens objective). Such components areoptionally organized upon a framework and/or enclosed within a housingstructure. It will be appreciated that the illustrations herein are ofexemplary embodiments and are not necessarily to be taken as limiting.Thus, for example, different embodiments can employ different placementof components relative to one another (e.g., embodiment A includes aheater/cooler as in FIG. 17, while embodiment B includes a heater/coolercomponent beneath its flow cell, etc.).

FIG. 18 presents an exemplary arrangement of a system that may be usedwith electrode-containing flow cells as disclosed herein. As can beseen, the system can be divided into certain basic groupings, e.g., area1800 comprising fluidics and reagent storage (including pumps and motorsor the like for producing and regulating fluid flow, heaters/coolers forproper reagent temperatures, etc.), area 1810 comprising flow cell anddetection (including one or more cameras or similar devices, one or morelasers or other light sources, one or more appropriate optical filtersand lenses, a temperature control actuator, e.g., with Peltierheating/cooling for control of the temperature conditions of the flowcell, a movable staging platform and motors controlling such tocorrectly position the various devices/components within the system),and area 1820 containing a computer module (including memory and a userinterface such as a display panel and keyboard, etc.).

As indicated above, the disclosed embodiments include systems anddevices for sequencing nucleic acids. Sequencing of a target fragmentmeans that a read of the chronological order of bases is established.The bases that are read do not need to be contiguous, although this isoften the case, nor does every base on the entire fragment have to besequenced during the sequencing. As explained, sequencing can be carriedout using any suitable sequencing technique, where nucleotides oroligonucleotides are added successively to a free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the nucleotide added is typically determinedafter each nucleotide addition. Sequencing techniques using sequencingby ligation, wherein not every contiguous base is sequenced, andtechniques such as massively parallel signature sequencing (MPSS) wherebases are removed from, rather than added to, the strands on the surfaceare also amenable to use with the systems and devices such as depictedin FIGS. 17 and 18.

In particular uses of the systems/devices herein, the flow cellscontaining the nucleic acid samples for sequencing are placed within theappropriate flow cell holder of the disclosed embodiments. The samplesfor sequencing can take the form of single molecules, amplified singlemolecules in the form of clusters, or beads comprising molecules ofnucleic acid. The nucleic acids are prepared such that they comprise anoligonucleotide primer adjacent to an unknown target sequence. Toinitiate the first SBS sequencing cycle, one or more differently labelednucleotides, and DNA polymerase, etc., are flowed into/through the flowcell by the fluid flow subsystem (various embodiments of which aredescribed herein). Either a single nucleotide can be added at a time, orthe nucleotides used in the sequencing procedure can be speciallydesigned to possess a reversible termination property, thus allowingeach cycle of the sequencing reaction to occur simultaneously in thepresence of all four labeled nucleotides (A, C, T, G). Where the fournucleotides are mixed together, the polymerase is able to select thecorrect base to incorporate and each sequence is extended by a singlebase. In such methods of using the systems of the disclosed embodiments,the natural competition between all four alternatives leads to higheraccuracy than wherein only one nucleotide is present in the reactionmixture (where most of the sequences are therefore not exposed to thecorrect nucleotide). Sequences where a particular base is repeated oneafter another (e.g., homopolymers) are addressed like any other sequenceand with high accuracy.

Fluid Flow

The fluid flow subsystem also flows the appropriate reagents to removethe blocked 3′ terminus (if appropriate) and the fluorophore from eachincorporated base. The substrate can be exposed either to a second roundof the four blocked nucleotides, or optionally to a second round with adifferent individual nucleotide. Such cycles are then repeated and thesequence of each cluster is read over the multiple chemistry cycles. Thecomputer aspect of the disclosed embodiments can optionally align thesequence data gathered from each single molecule, cluster or bead todetermine the sequence of longer polymers, etc. Alternatively, the imageprocessing and alignment can be performed on a separate computer.

In the various embodiments herein, the reagents, buffers, etc. used inthe sequencing of the nucleic acids are regulated and dispensed via afluid flow subsystem or aspect. FIGS. 19A-C present generalized diagramsof exemplary fluid flow arrangements of the disclosed embodiments, setup in one way push, eight way pull, and one way pull configurationsrespectively. In general, the fluid flow subsystem transports theappropriate reagents (e.g., enzymes, buffers, dyes, nucleotides, etc.)at the appropriate rate and optionally at the appropriate temperature,from reagent storage areas (e.g., bottles, or other storage containers)through the flow cell and optionally to a waste receiving area.

The fluid flow aspect is optionally computer controlled and canoptionally control the temperature of the various reagent components.For example, certain components are optionally held at cooledtemperatures such as 4° C.+1-1° C. (e.g., for enzyme containingsolutions), while other reagents are optionally held at elevatedtemperatures (e.g., buffers to be flowed through the flow cell when aparticular enzymatic reaction is occurring at the elevated temperature).

In some embodiments, various solutions are optionally mixed prior toflow through the flow cell (e.g., a concentrated buffer mixed with adiluent, appropriate nucleotides, etc.). Such mixing and regulation isalso optionally controlled by the fluid flow aspect of the disclosedembodiments. It is advantageous if the distance between the mixed fluidsand the flow cell is minimized in many embodiments. Therefore the pumpcan be placed after the flow cell and used to pull the reagents into theflow cell (FIGS. 19B and 19C) as opposed to having the pump push thereagents into the flow cell (as in FIG. 19A). Such pull configurationsmean that any materials trapped in dead volumes within the pump do notcontaminate the flow cell. The pump can be a syringe type pump, and canbe configured to have one syringe per flow channel to ensure even flowthrough each channel of the flow cell. The pump can be an 8 way pump, ifit is desired to use an 8 way flow cell, such as for example a Kloehn 8way syringe pump (Kloehn, Las Vegas, Nev.). A fluidics diagram of an 8way pull configuration is shown in FIG. 16B. In FIG. 16A, fluidicreagents are stored in reagent containers 1900 (e.g., buffers at roomtemperature, 5×SSC buffer, enzymology buffer, water, cleavage buffer,etc.) and 1910 (e.g., cooled containers for enzymes, enzyme mixes,water, scanning mix, etc.). Pump 1930 moves the fluids from the reagentcontainers through reagent valve 1940, priming/waste valve 1970 andinto/through flow cell 1960.

In FIG. 19B, fluidic reagents are stored in reagent containers 1902(e.g., buffers at room temperature similar to those listed above) and1903 (e.g., cooled containers for enzymes, etc. similar to those listedabove), linked through reagent valve 1901. Those of skill in the artwill be familiar with multi-way valves (such as the reagent valves) usedto allow controllable access of/to multiple lines/containers. Thereagent valve is linked into flow cell 1905 via an optional primingvalve (or waste valve) 1904, connected to optional priming pump 1906.The priming pump can optionally draw reagents from the containers upthrough the tubing so that the reagents are “ready to go” into the flowcell. Thus, dead air, reagents at the wrong temperature (e.g., becauseof sitting in tubing), etc. will be avoided. When the priming pump isdrawing, the outflow is shunted into the waste area. During non-priminguse, the reagents can be pulled through the flow cell using 8 channelpump 1907, which is connect to waste reservoir 1908.

In either embodiment (push or pull), the fluidic configurations cancomprise “sipper” tubes or the like that extend into the various reagentcontainers in order to extract the reagents from the containers. FIG.19C shows a single channel pump rather than an 8 channel pump. Singlechannel pump 1926 can also act as the optional priming pump, and thusoptional priming pump or waste valve 1923 can be connected directly topump 1926 through bypass 1925. The arrangement of components is similarin this embodiment as to that of FIG. 16B. Thus it comprises reagentcontainers 1921 and 1922, multi-way selector valve 1920, flow cell 1924,etc.

The fluid flow itself is optionally driven by any of a number of pumptypes, (e.g., positive/negative displacement, vacuum, peristaltic, etc.)such as an Encynova® 2-1 Pump (Encynova, Greeley, Colo.) or a Kloehn® V3Syringe Pump (Kloehn, Las Vegas, Nev.). Again, it will be appreciatedthat specific recitation of particular pumps, etc. herein should not betaken as necessarily limiting and that various embodiments can comprisedifferent pumps and/or pump types than those listed herein. In certainembodiments, the fluid delivery rate is from about 50 uL to about 500uL/min (e.g., controlled+/−2 uL) for the 8 channels. In the 8 way pullconfiguration, the flow can be between 10-100 uL/min/channel, dependingon the process. In some embodiments, the maximum volume of nucleotidereagents required for sequencing a polynucleotide of 25 bases is about12 mL.

Whichever pump or pump type is used herein, the reagents are optionallytransported from their storage areas to the flow cell through tubing.Such tubing, such as PTFE, can be chosen in order to, e.g., minimizeinteraction with the reagents. The diameter of the tubing can varybetween embodiments (and/or optionally between different reagent storageareas), but can be chosen based on, e.g., the desire to decrease “deadvolume” or the amount of fluid left in the lines; Furthermore, the sizeof the tubing can optionally vary from one area of a flow path toanother. For example, the tube size from a reagent storage area can beof a different diameter than the size of the tube from the pump to theflow cell, etc.

The fluid flow subsystem of the disclosed embodiments also can controlthe flow rate of the reagents involved. The flow rate is optionallyadjustable for each flow path (e.g., some flow paths can proceed athigher flow rates than others; flow rates can optionally be reversed;different channels can receive different reagent flows or differenttimings of reagent flows, etc.). The flow rate can be set in conjunctionwith the tube diameter for each flow path in order to have the propervolume of reagent, etc in the flow cell at a given time. For example, insome embodiments, the tubing through which the reagents flow is 0.3 mmID, 0.5 mm, or 1.0 mm while the flow rate is 480 uL/min or 120 uL/min.In some embodiments, the speed of flow is optionally balanced tooptimize the reactions of interest. High flow can cause efficientclearing of the lines and minimize the time spent in changing thereagents in a given flow cell volume, but can also cause a higher levelof shear flow at the substrate surface and can cause a greater problemwith leaks or bubbles. A typical flow rate for the introduction ofreagents can be 15 uL/min/channel in some embodiments.

The system can be further equipped with pressure sensors thatautomatically detect and report features of the fluidic performance ofthe system, such as leaks, blockages and flow volumes. Such pressure orflow sensors can be useful in instrument maintenance andtroubleshooting. The fluidic system can be controlled by the one or morecomputer component, e.g., as described below. It will be appreciatedthat the fluid flow configurations in the various embodiments of thedisclosed embodiments can vary, e.g., in terms of number of reagentcontainers, tubing length/diameter/composition, types of selector valvesand pumps, etc.

Heating/Cooling

The heating/cooling components of the system regulate the reactionconditions within the flow cell channels and reagent storageareas/containers (and optionally the camera, optics, and/or othercomponents), while the fluid flow components allow the substrate surfaceto be exposed to suitable reagents for incorporation (e.g., theappropriate fluorescently labeled nucleotides to be incorporated) whileunincorporated reagents are rinsed away. An optional movable stage uponwhich the flow cell is placed allows the flow cell to be brought intoproper orientation for laser (or other light) excitation of thesubstrate and optionally moved in relation to a lens objective to allowreading of different areas of the substrate. Additionally, othercomponents of the system are also optionally movable/adjustable (e.g.,the camera, the lens objective, the heater/cooler, etc.). During laserexcitation, the image/location of emitted fluorescence from the nucleicacids on the substrate is captured by the camera component, thereby,recording the identity, in the computer component, of the first base foreach single molecule, cluster or bead.

Microcontroller

The existing system may be modified to incorporate separate logic thatcontrols activation of the different electrodes at different times,associated with delivery of different libraries to the flow cell. Thismay be performed by a microcontroller that interfaces with the flow celland the sequencing system logic. The microcontroller could be utilizedduring capture of polynucleotides delivered into the flow cell. Inanother embodiment, the microcontroller may be embedded in the systemitself.

FIG. 20 shows how the electrodes of the flow cell may be interfaced withthe sequencing system and controller. In some embodiments, the recipefor cluster generation may include desired locations for hybridization,and extra hardware (FPGA, DAC, ADC) may be added to the controller boardto read desired spatial hybridization statuses and control eachelectrode accordingly. In some embodiments, these features may beimplemented on an Illumina® cBot®/MiSeq® controller board. Themicrocontroller may be designed or programmed to operate the electrodesin direct current (DC) or alternating current (AC) mode. In variousembodiments, the controller synchronizes delivery of particularlibraries (fluidically to a flow cell) with activation of certainelectrodes in the library capture region.

EXAMPLES Electrode Configuration: Interdigitated Electrode Configuration

In one example, electrodes were arranged such that interdigitated goldelectrodes are arranged in parallel to each other. Multiple electrodescan be arranged in parallel to each other on one flow surface with thecounter electrodes on the opposite flow cell surface. Alternatively,multiple electrodes can be arranged on both surfaces as well. The flowcells used to generate the data in some of the examples described hereinhave dimensions of 5.8 mm×5.8 mm×100 um.

FIG. 21 depicts an example of this configuration, where one set ofelectrodes 2102 was controlled by voltage source V1 and another set ofelectrodes 2104 was controlled by voltage source V2.

In the example of FIG. 22, two libraries were sequentially loaded ontothe library capture surface while activating a different set ofelectrodes, such that library 1 was localized on the electrodescontrolled by V1 and library 2 was localized on the electrodescontrolled by V2. After a library was loaded onto the capture surface,the capture surface was washed to remove unbound polynucleotides. Afterboth libraries were loaded, the hybridized polynucleotides underwentbridge amplification and linearization and were subsequently sequencedby synthesis. FIG. 22 demonstrates that subsequent sequencing revealedthat the different libraries were localized to different electrodes.

Some embodiments include a method of sequencing a polynucleotide sample,the method comprising:

(a) providing a solid support having a plurality of electrodes disposedthereon, the solid support including a polymer layer over the pluralityof electrodes, the polymer layer including a plurality of forward andreverse amplification primers immobilized thereon, the solid supportalso including electrical leads connected to the plurality of electrodespermitting the electrodes to be independently addressable, the solidsupport also including a fluid direction system for controllablydelivering a plurality of polynucleotide libraries through the solidsupport at different time periods;

(b) applying a positive charge to a first electrode of the plurality ofelectrodes;

(c) delivering a first polynucleotide library along the solid supportsuch that polynucleotides from the first polynucleotide library areattracted to forward and reverse amplification primers disposedproximate the first electrode such that members of the firstpolynucleotide library hybridize to forward and reverse amplificationprimers proximate the first electrode, wherein members of the firstpolynucleotide library do not substantially hybridize to forward andreverse amplification primers located proximate electrodes that do nothave positive charge applied;

(d) applying a positive charge to a second electrode of the plurality ofelectrodes; and

(e) delivering a second polynucleotide library along the solid supportsuch that polynucleotides from the second polynucleotide library areattracted to forward and reverse amplification primers disposedproximate the second electrode such that members of the secondpolynucleotide library hybridize to forward and reverse amplificationprimers proximate the second electrode, wherein members of the secondpolynucleotide library do not substantially hybridize to forward andreverse amplification primers located proximate electrodes that do nothave positive charge applied.

What is claimed:
 1. A method of sequencing, the method comprising:introducing a first nucleic acid library to a library sequencing regionof a flow cell comprising: a substrate having an inner surface facingthe library sequencing region, a plurality of forward and reverseamplification primers disposed over the inner surface and providing anucleic acid library capture surface of the library sequencing region,and a plurality of electrodes disposed along the substrate proximate thelibrary capture surface; applying a positive charge to a first set ofone or more electrodes, from the plurality of electrodes, while thefirst nucleic acid library flows through the library sequencing regionto thereby attract nucleic acids from the first nucleic acid library tothe forward and reverse amplification primers disposed proximate thefirst set of one or more electrodes such that members of the firstnucleic acid library hybridize to forward and reverse amplificationprimers proximate the first set of one or more electrodes, whereinmembers of the first nucleic acid library do not substantially hybridizeto forward and reverse amplification primers located proximateelectrodes that do not have positive charge applied; introducing asecond nucleic acid library to a library sequencing region flow cell;and applying a positive charge to a second set of one or moreelectrodes, from the plurality of electrodes, while the second nucleicacid library flows through the library sequencing region to therebyattract nucleic acids from the second nucleic acid library to theforward and reverse amplification primers disposed proximate the secondset of one or more electrodes such that members of the second nucleicacid library hybridize to forward and reverse amplification primersproximate the second set of one or more electrodes, wherein members ofthe second nucleic acid library do not substantially hybridize toforward and reverse amplification primers located proximate electrodesthat do not have positive charge applied.
 2. The method of claim 1,further comprising one or both of: applying a negative charge toelectrodes adjacent to the first set of electrodes while applying thepositive charge to the first set of electrodes; and applying a negativecharge to electrodes adjacent to the second set of electrodes whileapplying the positive charge to the second set of electrodes.
 3. Themethod of claim 1, wherein introducing the first or second nucleic acidlibrary comprises flowing the first or second nucleic acid library in asolution comprising one or more protective reagents that undergoes aredox reaction at an electric potential that is below the electricpotential at which water electrolyzes, and wherein the redox reactionproduces only products that are substantially benign to nucleic acids.4. The method of claim 43, wherein said one or more protective reagentsare selected from the group consisting of α-thioglycerol,dithiothreitol, hydroquinone, ferrocyanide, and β-mercaptoethanol. 5.The method of claim 1, further comprising preparing the first nucleicacid library by: fragmenting a complex polynucleotide sample to generatea plurality of target polynucleotide fragments; and ligating identicalmismatched adapter polynucleotides to both ends of each of the differenttarget polynucleotide fragments to form adapter-target constructs,wherein each mismatched adapter is formed from two annealedpolynucleotide strands that form a bimolecular complex comprising atleast one double-stranded region and a mismatched region comprisingportions of both strands, wherein the ligating covalently attaches eachstrand of the at least one double-stranded region to each respectivestrand of each of the different target polynucleotide fragment togenerate adapter-target constructs comprising covalently attached 5′ and3′ adapter sequences.
 6. The method of claim 1, wherein each hybridizednucleic acid is amplified by: forming at least one nucleic acid templatecomprising the at least one nucleic acid to be amplified, wherein the atleast one nucleic acid contains an oligonucleotide sequence Y at the 5′end and an oligonucleotide sequence Z at the 3′ end, and the at leastone nucleic acid carries a means for immobilizing the at least onenucleic acid to a solid support at the 5′ end; mixing the at least onenucleic acid template, in the presence of the solid support, with one ormore colony primers X, each of which can hybridize to theoligonucleotide sequence Z and carries a means for immobilizing thecolony primer to the solid support at the 5′ end, whereby the 5′ ends ofboth the at least one nucleic acid template and the colony primers areimmobilized to the solid support, wherein said 5′ ends of both the atleast one nucleic acid template and the colony primers are immobilizedto said solid support such that they cannot be removed by washing withwater or aqueous buffer under DNA denaturing conditions; and performingone or more nucleic acid amplification reactions on the immobilizednucleic acid template, so that nucleic acid colonies are generated. 7.The method of claim 6, the method further comprising: moving one or morefluorescently labeled reagents through the flow cell into contact withthe hybridized members of the first and second nucleic acid libraries,wherein the reagents comprise components to extend a second sequencecomplementary to the hybridized polynucleotides; illuminating thehybridized polynucleotides with at least one excitation laser coupledthrough a fiberoptic device; detecting, using at least onecharge-coupled device (CCD) camera, fluorescence emissions of thefluorescently labeled reagents; and determining, based on thefluorescence emissions, an identity of the second sequence.
 8. Themethod of claim 1, wherein the forward and reverse amplification primersare to hybridize specific gene sequences, wherein the specific genesequences include a barcode region of the nucleic acid libraries, anadapter region of the nucleic acid libraries, or nucleic acid sequencesof interest within the nucleic acid libraries.
 9. A system forsequencing, the system comprising: a solid support having a plurality ofelectrodes disposed thereon, the solid support including an attachmentlayer over the plurality of electrodes, the attachment layer having alibrary capture surface including a plurality of forward and reverseamplification primers immobilized thereon; electrical leads connected tothe plurality of electrodes to permit the electrodes to be independentlyaddressable; a fluid direction system for controllably delivering aplurality of polynucleotide libraries in a buffer with a reducing agentto the library capture surface during different time periods; and acontroller for controlling the fluid direction system and for deliveringcurrent and/or potential to the electrodes, wherein the controller isto: apply a positive charge to a first set of one or more of theplurality of electrodes while the fluid direction system delivers afirst polynucleotide library along the solid support such thatpolynucleotides from the first polynucleotide library are attracted toforward and reverse amplification primers disposed proximate the firstset of electrodes such that members of the first polynucleotide libraryhybridize to forward and reverse amplification primers proximate thefirst set of electrodes, wherein members of the first polynucleotidelibrary do not substantially hybridize to forward and reverseamplification primers located proximate electrodes that do not havepositive charge applied, and apply a positive charge to a second set ofone or more of the electrodes while the fluid direction system deliversa second polynucleotide library along the solid support such thatpolynucleotides from the second polynucleotide library are attracted toforward and reverse amplification primers disposed proximate the secondelectrode such that members of the second polynucleotide libraryhybridize to forward and reverse amplification primers proximate thesecond set of electrodes, wherein members of the second polynucleotidelibrary do not substantially hybridize to forward and reverseamplification primers located proximate electrodes that do not havepositive charge applied.
 10. The system of claim 9, wherein thecontroller is further to cause the fluid direction system to deliver thepolynucleotide libraries to distinct lanes of the flow cell.
 11. Thesystem of claim 9, wherein the controller is to apply the positivecharge to the set of first or second electrodes that is at a voltage inthe range of approximately 0.5-3V, produces a current in the range ofapproximately 250 nA-5 tA, or produces an electric field in the range ofapproximately 10-200 V/cm.
 12. The system of claim 9, where theplurality of electrodes comprises a conductor selected from the groupconsisting of gold, indium-doped tin oxide (ITO), silver, tin, titanium,copper, platinum, palladium, polysilicon, and carbon.